Amino acid transporters and uses

ABSTRACT

The present invention relates to novel mammalian amino acid transporter proteins and the genes that encode such proteins. The invention is directed toward the isolation, characterization and pharmacological use of a human amino acid transporter protein termed EAAT4 and genes encoding such a transporter. The invention specifically provides isolated complementary DNA copies of mRNA corresponding to this transporter gene. Also provided are recombinant expression constructs capable of expressing this amino acid transporter gene in cultures of transformed prokaryotic and eukaryotic cells, as well as such cultures of transformed cells that synthesize the human amino acid transporter protein encoded therein. The invention also provides methods for screening in vitro compounds having transport-modulating properties using preparations of transporter proteins from such cultures of cells transformed with recombinant expression constructs.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/140,729, filed Oct. 20, 1993, now U.S. Pat. No. ______ , issued______, 1996, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under NationalInstitute of Health grant DA07595. The government has certain rights tothis invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to amino acid transporters from mammalianspecies and the genes corresponding to such transporters. Specifically,the invention relates to the isolation, cloning and sequencing ofcomplementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novelhuman amino acid transporter gene. The invention also relates to theconstruction of recombinant expression constructs comprising such cDNAsfrom a novel human amino acid transporter gene of the invention, saidrecombinant expression constructs being capable of expressing amino acidtransporter protein in cultures of transformed prokaryotic andeukaryotic cells as well as in amphibian oocytes. Production of thetransporter protein of the invention in such cultures and oocytes isalso provided. The invention relates to the use of such cultures of suchtransformed cells to produce homogeneous compositions of the noveltransporter protein. The invention also provides cultures of such cellsand oocytes expressing transporter protein for the characterization ofnovel and useful drugs. Antibodies against and epitopes of thetransporter protein are also provided by the invention.

[0005] 2. Background of the Invention

[0006] The approximately 20 naturally-occurring amino acids are thebasic building blocks for protein biosynthesis. Certain amino acids,such as glutamate and glycine, as well as amino acid derivatives such asγ-aminobutyric acid (GABA), epinephrine and norepinephrine, andhistamine, are also used as signaling molecules in higher organisms suchas man. For these reasons, specialized trans-membrane transporterproteins have evolved in all organisms to recover or scavengeextracellular amino acids (see Christensen, 1990, Physiol Rev. 10: 43-77for review).

[0007] These transporter proteins play a particularly important role inuptake of extracellular amino acids in the vertebrate brain (seeNicholls & Attwell, 1990, TiPS 11: 462-468). Amino acids that functionas neurotransmitters must be scavenged from the synaptic cleft betweenneurons to enable continuous repetitive synaptic transmission. Moreimportantly, it has been found that high extracellular concentrations ofcertain amino acids (including glutamate and cysteine) can causeneuronal cell death. High extracellular amino acid concentrations areassociated with a number of pathological conditions, including ischemia,anoxia and hypoglycemia, as well as chronic illnesses such asHuntington's disease, Parkinson's disease, Alzheimer's disease, epilepsyand amyotrophic lateral sclerosis (ALS; see Pines et al., 1992, Nature360: 464-467).

[0008] Glutamate is one example of such an amino acid. Glutamate is anexcitatory neurotransmitter (i.e., excitatory neurons use glutamate as aneurotransmitter). When present in excess (>about 300 μM; Bouvier etal., 1992, Nature 360: 471-474; Nicholls & Attwell, ibid.; >5 μM for 5min.; Choi et al., 1987, J. Neurosci. 7: 357-358), extracellularglutamate causes neuronal cell death. Glutamate transporters play apivotal role in maintaining non-toxic extracellular concentrations ofglutamate in the brain. During anoxic conditions (such as occur duringischemia), the amount of extracellular glutamate in the brain risesdramatically. This is in part due to the fact that, under anoxicconditions, glutamate transporters work in reverse, thereby increasingrather than decreasing the amount of extracellular glutamate found inthe brain. The resultingly high extracellular concentration of glutamatecauses neuron death, with extremely deleterious consequences for motorand other brain functions, resulting in stroke and other instances oforganic brain dysfunction.

[0009] This important role for amino acid transporters in maintainingbrain homeostasis of extracellular amino acid concentrations hasprovided the impetus for the search for and development of compounds tomodulate and control transporter function. However, conventionalscreening methods require the use of animal brain slices in bindingassays as a first step. This is suboptimal for a number of reasons,including interference in the binding assay by non-specific binding ofheterologous (i.e., non-transporter) cell surface proteins expressed bybrain cells in such slices; differential binding by cells other thanneuronal cells present in the brain slice, such as glial cells or bloodcells; and the possibility that putative drug binding behavior in animalbrain cells will differ from the binding behavior in human brain cellsin subtle but critical ways. The ability to synthesize human transportermolecules in vitro would provide an efficient and economical means forrational drug design and rapid screening of potentially usefulcompounds.

[0010] Amino acid transporters are known in the art, and some of theseproteins have been isolated biochemically and their corresponding geneshave been recently cloned using genetic engineering means.

[0011] Christensen et al., 1967, J. Biol. Chem. 242: 5237-5246 reportthe discovery of a neutral amino acid transporter (termed the ACStransporter) in Erlich ascites tumor cells.

[0012] Makowske & Christensen, 1982, J. Biol. Chem. 257: 14635-14638provide a biochemical characterization of hepatic amino acid transport.

[0013] Kanner & Schuldiner, 1987, CRC Crit. Rev. Biochem. 22: 1-38provide a review of the biochemistry of neurotransmitters.

[0014] Olney et al., 1990, Science 248: 596-599 disclose that the aminoacid cysteine is a neurotoxin when present in excess extracellularly.

[0015] Wallace et al., 1990, J. Bacteriol. 172: 3214-3220 report thecloning and sequencing of a glutamate/aspartate transporter gene termedgltP from Escherichia coli strain K12.

[0016] Kim et al., 1991, Nature 352: 725-728 report the discovery that acationic amino acid transporter is the cell surface target for infectionby ecotropic retroviruses in mice.

[0017] Wang et al., 1991, Nature 352: 729-731 report the discovery thata cationic amino acid transporter is the cell surface target forinfection by ecotropic retroviruses in mice.

[0018] Maenz et al., 1992, J. Biol. Chem. 267: 1510-1516 provide abiochemical characterization of amino acid transport in rabbit jejunalbrush border membranes.

[0019] Bussolati et al., 1992, J. Biol. Chem. 267: 8330-8335 report thatthe ASC transporter acts in an electrochemically neutral manner so thatsodium ion co-transport occurs without disrupting the normal membranepotential of the cells expressing the transporter.

[0020] Engelke et al., 1992, J. Bacteriol. 171: 5551-5560 report thecloning of a dicarboxylate carrier from Rhizobium meliloti.

[0021] Guastella et al., 1992, Proc. Natl. Acad. Sci. USA 89: 7189-7193disclose the cloning of a sodium ion and chloride ion-dependent glycinetransporter from a glioma cell line that is expressed in the ratforebrain and cerebellum.

[0022] Kavanaugh et al., 1992, J. Biol. Chem. 267:22007-22009 reportthat biochemical characterization of a rat brain GABA transporterexpressed in vitro in Xenopus laevis oocytes.

[0023] Storck et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10955-10959disclose the cloning and sequencing of a sodium ion-dependentglutamate/aspartate transporter from rat brain termed GLAST1.

[0024] Bouvier et al., ibid., disclose the biochemical characterizationof a glial cell-derived glutamate transporter.

[0025] Pines et al., ibid., report the cloning and sequencing of a glialcell glutamate transporter from rat brain termed GLT-1.

[0026] Kanai & Hediger, 1992, Nature 360: 467-471 disclose the cloningand sequencing of a sodium ion-dependent, high affinity glutamatetransporter from rabbit small intestine termed EAAC1.

[0027] Arriza et al., 1992, J. Biol. Chem. 268: 15329-15332 disclose agene for a novel neutral amino acid transporter.

[0028] Kong et al., 1993, J. Biol. Chem. 268: 1509-1512 report thecloning and sequencing of a sodium-ion dependent neutral amino acidtransporter of the A type that is homologous to a sodium-ion dependentglucose transporter.

[0029] Arriza et al., 1994, J. Neurosci. 14: 5559-5569 disclose genesfor three novel glutamate transporters.

[0030] Nicholls & Attwell, ibid., review the role of amino acids andamino acid transporters in normal and pathological brain functions.

SUMMARY OF THE INVENTION

[0031] The present invention relates to the cloning, expression andfunctional characterization of mammalian amino acid transporter genes.The invention comprises nucleic acids having a nucleotide sequence of anovel amino acid transporter gene. The nucleic acids provided by theinvention each comprise a complementary DNA (cDNA) copy of thecorresponding mRNA transcribed in vivo from the amino acid transportergene of the invention. Also provided is the deduced amino acid sequencesof the cognate protein of the cDNA provided by the invention.

[0032] This invention provides nucleic acids, nucleic acid hybridizationprobes, recombinant eukaryotic expression constructs capable ofexpressing the amino acid transporter of the invention in cultures oftransformed cells and in amphibian oocytes, such cultures of transformedeukaryotic cells and such amphibian oocytes that synthesize the aminoacid transporter of the invention, a homogeneous composition of theamino acid transporter protein, and antibodies against and epitopes ofthe amino acid transporter protein of the invention. Methods forcharacterizing this transporter protein and methods for using thisprotein and cells and oocytes expressing this protein for thedevelopment of agents having pharmacological uses related to thistransporter protein are also provided by the invention.

[0033] In a first aspect, the invention provides a nucleic acid having anucleotide sequence encoding a human excitatory amino acid transporterthat is the EAAT4 transporter (SEQ ID No:1). In this embodiment of theinvention, the nucleotide sequence includes 1734 nucleotides of thehuman EAAT4 cDNA comprising 1692 nucleotides of coding sequence, 8nucleotides of 5′ untranslated sequence and 34 nucleotides of 3′untranslated sequence. In this embodiment of the invention, thenucleotide sequence of the EAAT4 transporter is the nucleotide sequencedepicted in FIG. 1 (SEQ ID No:1).

[0034] In another aspect, the invention comprises a homogeneouscomposition of the 61.6 kilodalton (kD) mammalian EAAT4 transporter andderivatives thereof, said size being understood to be the size of theprotein before any post-translational modifications thereof. The aminoacid sequence of the EAAT4 transporter and derivatives thereofpreferably is the amino acid sequence of the human EAAT4 transporterprotein shown in FIG. 2 (SEQ ID No:2). EAAT4 protein molecules providedby the invention are understood to have substantially the samebiological properties as the EAAT4 protein molecule encoded by thenucleotide sequence described herein.

[0035] This invention provides both nucleotide and amino acid probesderived from the sequences herein provided. The invention includesprobes isolated from either cDNA or genomic DNA, as well as probes madesynthetically with the sequence information derived therefrom. Theinvention specifically includes but is not limited to oligonucleotide,nick-translated, random primed, or in vitro amplified probes made usingcDNA or genomic clone embodying the invention, and oligonucleotide andother synthetic probes synthesized chemically using the nucleotidesequence information of cDNA or genomic clone embodiments of theinvention.

[0036] It is a further object of this invention to provide such nucleicacid hybridization probes to determine the pattern, amount and extent ofexpression of this transporter gene in various tissues of mammals,including humans. It is also an object of the present invention toprovide nucleic acid hybridization probes derived from the sequences ofthe amino acid transporter gene of the invention to be used for thedetection and diagnosis of genetic diseases. It is an object of thisinvention to provide nucleic acid hybridization probes derived from theDNA sequence of the amino acid transporter gene herein disclosed to beused for the detection of novel related receptor genes.

[0037] The present invention also includes synthetic peptides made usingthe nucleotide sequence information comprising the cDNA embodiments ofthe invention. The invention includes either naturally occurring orsynthetic peptides which may be used as antigens for the production ofamino acid transporter-specific antibodies, or used for competitors ofamino acid transporter molecules for amino acid, agonist, antagonist ordrug binding, or to be used for the production of inhibitors of thebinding of agonists or antagonists or analogues thereof to such aminoacid transporter molecules.

[0038] The present invention also provides antibodies against andepitopes of the mammalian amino acid transporter molecules of theinvention. It is an object of the present invention to provideantibodies that are immunologically reactive to the amino acidtransporter of the invention. It is a particular object to providemonoclonal antibodies against this amino acid transporter, mostpreferably the human excitatory amino acid transporter as hereindisclosed. Hybridoma cell lines producing such antibodies are alsoobjects of the invention. It is envisioned that such hybridoma celllines may be produced as the result of fusion between anon-immunoglobulin producing mouse myeloma cell line and spleen cellsderived from a mouse immunized with a cell line which expresses antigensor epitopes of an amino acid transporter of the invention. The presentinvention also provides hybridoma cell lines that produce suchantibodies, and can be injected into a living mouse to provide anascites fluid from the mouse that is comprised of such antibodies. It isa further object of the invention to provide immunologically-activeepitopes of the amino acid transporter of the invention. Chimericantibodies immunologically reactive against the amino acid transporterprotein of the invention are also within the scope of this invention.

[0039] The present invention provides recombinant expression constructscomprising a nucleic acid encoding an amino acid transporter of theinvention wherein the construct is capable of expressing the encodedamino acid transporter in cultures of cells or amphibian oocytestransformed with the construct. Preferred embodiments of such constructscomprise the human EAAT4 cDNA (SEQ ID No.:1), the construct beingcapable of expressing the amino acid transporter encoded therein incells and oocytes transformed with the construct or into which theconstruct has otherwise been introduced.

[0040] The invention also provides cultures cells transformed with therecombinant expression constructs of the invention, each such culturesbeing capable of and in fact expressing the amino acid transporterencoded in the transforming construct. The invention also providesamphibian oocytes into which a recombinant expression construct of theinvention is introduced, each such oocyte being capable of and in factexpressing the amino acid transporter encoded in the transformingconstruct.

[0041] The present invention also includes within its scope proteinpreparations of prokaryotic and eukaryotic cell membranes containing theamino acid transporter protein of the invention, derived from culturesof prokaryotic or eukaryotic cells, respectively, transformed with therecombinant expression constructs of the invention. In a preferredembodiment, such preparations of cell membranes comprise the amino acidtransporter protein of the invention.

[0042] The invention also provides methods for screening compounds fortheir ability to inhibit, facilitate or modulate the biochemicalactivity of the amino acid transporter molecules of the invention, foruse in the in vitro screening of novel agonist and antagonist compounds.In preferred embodiments, cells, particularly amphibian oocytestransformed with a recombinant expression construct of the invention arecontacted with such a compound, and the effect of the compound on thetransport of the appropriate amino acid is assayed. Additional preferredembodiments comprise quantitative analyses of such effects. Alsoprovided are assays that distinguish between the effect of suchcompounds on amino acid transport from effects of such compounds onchloride ion transport by the transporters of the invention.

[0043] The present invention is also useful for the detection ofanalogues, agonists or antagonists, heretofore known or unknown, of theamino acid transporters of the invention, either naturally occurring orembodied as a drug. In preferred embodiments, such analogues, agonistsor antagonists may be detected in blood, saliva, semen, cerebrospinalfluid, plasma, lymph, or any other bodily fluid. In additional preferredembodiments, the invention provides methods for detecting andidentifying analogues, agonists or antagonists that preferentiallyaffect either the amino acid uptake function or the chloride ion channelfunction of the amino acid transporters of the invention.

[0044] Specific preferred embodiments of the present invention willbecome evident from the following more detailed description of certainpreferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 illustrates the nucleotide (SEQ ID No.:1) sequence of thehuman EAAT4 excitatory amino acid transporter.

[0046]FIG. 2 illustrates the amino acid (SEQ ID No.:2) sequence of thehuman EAAT4 excitatory amino acid transporter.

[0047]FIG. 3 presents an amino acid sequence comparison between humanEAAT4 and the previously-disclosed excitatory amino acid transportersEAAT1 (SEQ ID Nos.: 3 & 4), EAAT2 (SEQ ID Nos.: 5&6) and EAAT3 (SEQ IDNos.: 7&8).

[0048]FIG. 4 shows the pattern of expression of EAAT4 mRNA in humanbrain tissue; β-actin is shown as a control for the amount of RNA ineach lane.

[0049]FIG. 5 shows the pattern of expression of EAAT4 mRNA in humantissues; β-actin is shown as a control for amount of RNA in each lane.

[0050]FIG. 6A illustrates transmembrane electrochemical currents inXenopus laevis oocytes microinjected with RNA encoding EAAT4 cRNA andcontacted with 10 μM L-aspartate at varying voltage-clamped voltages.The magnitude of the induced current and the time course of inductionare shown with reference to the indicator bars in the upper right-handcorner of the Figure.

[0051]FIG. 6B is a graph showing the relationship between the magnitudeof the applied voltage clamp and the induced current in the presence ofincreasing amounts of gluconate ion in substitution for chloride ion.Open circles correspond to 104 mM chloride ion (no gluconate ionpresent), closed circles correspond to 56 mM chloride ion, open squarescorrespond to 20 mM chloride ion and closed squares correspond to 0 mMchloride ion (i.e., chloride ion is substituted entirely by gluconateion).

[0052]FIG. 6C is a semilogarithmic plot of the reversal potential versusthe log of the chloride ion concentration, showing that the applicationof excitatory amino acids to EAAT4 induces a large chloride ion current.

[0053]FIG. 6D illustrates transmembrane electrochemical currents inXenopus oocytes microinjected with RNA encoding EAAT4 cRNA and contactedwith 10 μM L-aspartate in the presence and absence of sodium ion in theperfusate. The magnitude of the induced current and the time course ofinduction are shown with reference to the indicator bars in the upperright-hand corner of the Figure.

[0054]FIG. 7A is a graph illustrating the current-voltage (I-V)relationship typical for EAAT1, EAAT2, and EAAT3, illustrated forEAAT-2. In this example, the application of L-glutamate to EAAT2, thereis inward rectification of the current, and outward currents are seenonly at membrane potentials more positive than +40 mV.

[0055]FIG. 7B is a graph showing the concentration and voltagedependence of L-aspartate induced currents in EAAT4. The current isdose-dependent, linear, and reverses (becomes outward) at a membranepotential of ˜−20 mV. L-aspartate concentrations shown are 0.3 μm(filled circles), 1 μM (open circles), 10 μM (open diamonds), and 100 μM(filled diamonds).

[0056]FIG. 7C is a graph comparing the current-voltage relationships ofL-aspartate and L-glutamate, for concentrations of 100 μM L-aspartate(filled circles) and 100 μM L-glutamate (open circles).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The term “human amino acid transporter EAAT4” as used hereinrefers to proteins having substantially the same biological activity asthe protein encoded by the nucleic acid depicted in FIG. 2 (SEQ IDNo.:1). This definition is intended to encompass allelic variations inthe EAAT4 sequence, either naturally occurring or the product of invitro chemical or genetic modification. Each such variant will beunderstood to have essentially the same nucleotide sequence as thenucleotide sequence of the corresponding EAAT4 disclosed herein. Clonednucleic acid provided by the present invention may encode EAAT4 proteinof any species of origin, including, for example, mouse, rat, rabbit,cat, and human, but preferably the nucleic acid provided by theinvention encodes EAAT4 receptors of mammalian, most preferably human,origin.

[0058] The term “excitatory amino acid” is intended to encompassnaturally-occurring and synthetic amino acids such as L-aspartate andL-glutamate, as well as homologues, analogues or derivatives thereof.The terms is also intended to encompass agonists, antagonists andinhibitors of mammalian glutamate receptors.

[0059] The term “detectably labeled” is intended to encompass anyreporter molecule capable of being detected by radiometric, fluorescent,spectrophotometric or other physical or chemical means. Particularexamples include radiolabels, including but not limited to ³H and ¹⁴C.

[0060] The term “chloride equilibrium potential” is intended to mean themembrane potentiat at which there is no detectable chloride ion fluxacross the cell membrane.

[0061] The nucleic acid hybridization probes provided by the inventioncomprise DNA or RNA having the nucleotide sequence of the amino acidtransporters, depicted in FIG. 1 (SEQ ID No.:1), or any portion thereofeffective in nucleic acid hybridization under stringency conditionssufficient to permit specific hybridization of the probe to acomplementary nucleic acid sequence. Mixtures of such nucleic acidhybridization probes are also within the scope of this embodiment of theinvention. Nucleic acid probes as provided herein are useful fordetecting novel amino acid transporter genes related to the EAAT4 genedisclosed herein, specifically including homologous or syntenictransporter genes in non-human mammalian species. Nucleic acid probes asprovided herein are also useful for detecting amino acid transportergene expression in cells and tissues using techniques well-known in theart, including but not limited to Northern blot hybridization, in situhybridization and Southern hybridization to reversetranscriptase-polymerase chain reaction (RT-PCR) product DNAs. Theprobes provided by the present invention, including oligonucleotidesprobes derived therefrom, are useful are also useful for Southernhybridization of mammalian, preferably human, genomic DNA for screeningfor restriction fragment length polymorphism (RFLP) associated withgenetic disorders.

[0062] The production of proteins such as these amino acid transportermolecules from cloned genes by genetic engineering means is well knownin this art. The discussion which follows is accordingly intended as anoverview of this field, and is not intended to reflect the full state ofthe art.

[0063] DNA encoding an amino acid transporter may be obtained, in viewof the instant disclosure, by chemical synthesis, by screening reversetranscripts of mRNA from appropriate cells or cell line cultures, byscreening genomic libraries from appropriate cells, or by combinationsof these procedures, as illustrated below. Screening of mRNA or genomicDNA may be carried out with oligonucleotide probes generated from thenucleic acid sequence information from each of the amino acidtransporters disclosed herein. Probes may be labeled with a detectablegroup such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with know procedures and used inconventional hybridization assays, as described in greater detail in theExamples below. In the alternative, amino acid transporter-derivednucleic acid sequences may be obtained by use of the polymerase chainreaction (PCR) procedure, using PCR oligonucleotide primerscorresponding to nucleic acid sequence information derived from an aminoacid transporter as provided herein. See U.S. Pat. Nos. 4,683,195 toMullis et al. and 4,683,202 to Mullis.

[0064] The amino acid transporter protein of the invention may besynthesized in host cells, in particular amphibian oocytes, transformedwith a recombinant expression construct comprising a nucleic acidencoding amino acid transporter cDNA. Such recombinant expressionconstructs can also be comprised of a vector that is a replicable DNAconstruct. Vectors are used herein either to amplify DNA encoding anamino acid transporter and/or to express DNA encoding an amino acidtransporter gene. For the purposes of this invention, a recombinantexpression construct is a replicable DNA construct in which a nucleicacid encoding an amino acid transporter is operably linked to suitablecontrol sequences capable of effecting the expression of the amino acidtransporter in a suitable host or host cell.

[0065] The need for such control sequences will vary depending upon thehost selected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter, optional or ancillarytranscription control sequences, such as transcription factor bindingdomains, enhancer sequences, and other eukaryotic “operator” sequencesto control transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences which control the termination oftranscription and translation. Amplification vectors do not requireexpression control domains. All that is needed is the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. See,Sambrook et al., 1990, Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Press: New York).

[0066] Vectors useful for practicing the present invention includeplasmids, viruses (including phage), retroviruses, and integratible DNAfragments (i.e., fragments integratible into the host genome byhomologous recombination). The vector replicates and functionsindependently of the host genome, or may, in some instances, integrateinto the genome itself. Suitable vectors will contain replicon andcontrol sequences which are derived from species compatible with theintended expression host. A preferred vector is pCMV5 (Andersson et al.,1989, J. Biol. Chem. 264: 8222 -8229). Transformed host cells are cellswhich have been transformed or transfected with recombinant expressionconstructs made using recombinant DNA techniques and comprising nucleicacid encoding an amino acid transporter protein. Preferred host cellsare Xenopus laevis oocytes, oocytes from other amphibian species, andCOS-7 cells (Gluzman, 1981, Cell 23: 175-182). Transformed host cellsmay express the amino acid transporter protein, but host cellstransformed for purposes of cloning or amplifying nucleic acidhybridization probe DNA need not express the transporter. Whenexpressed, the amino acid transporter protein molecules of the inventionwill typically be located in the host cell membrane. See, Sambrook etal., ibid.

[0067] Cultures of cells derived from multicellular organisms are adesirable host for recombinant amino acid transporter protein synthesis.In principal, any higher eukaryotic cell culture is useful, whether fromvertebrate or invertebrate culture. However, mammalian cells arepreferred, as illustrated in the Examples. Propagation of such cells incell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse & Patterson, editors (1973). Examples of usefulhost cell lines are human 293 cells, VERO and HeLa cells, Chinesehamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK celllines. COS-7 cells are preferred.

[0068] Certain other primary host cells, not subjected to prolongedtissue culture adaptation, can be used to produce the amino acidtransporters of the invention, particularly amphibian oocytes. Amphibianoocytes are useful for expressing the mammalian excitatory transportersof this invention, most preferably oocytes from Xenopus laevis or otheramphibian, which oocytes are used to provide cells convenient for thepractice of some of the inventive methods disclosed herein.

[0069] Thus, the invention also provides a method for making the humanEAAT4 amino acid transporters of the invention, and membranepreparations comprising this transporter, by introducing nucleic acidencoding the transporter into an appropriate prokaryotic, or preferably,eukaryotic, most preferably mammalian, cell that is capable ofexpressing the transporter protein.

[0070] The invention provides homogeneous compositions of the humanEAAT4 amino acid transporter proteins produced by transformed eukaryoticcells as provided herein. Such a homogeneous compositions are intendedto be comprised of the corresponding amino acid transporter protein thatcomprises at least 50-90% of the protein in such a homogenouscomposition. The invention also provides membrane preparations fromcells expressing the amino acid transporter protein as the result oftransformation with a recombinant expression construct, as describedherein.

[0071] Amino acid transporter protein made from cloned genes inaccordance with the present invention may be used for screening aminoacid analogues, or inhibitors, agonists or antagonists of amino acidtransport, or for determining the amount of such agonists or antagonistsin a solution of interest (e.g., blood plasma or serum). For example,host cells may be transformed with a recombinant expression construct ofthe present invention, an amino acid transporter expressed in those hostcells, and the cells or membranes thereof used to screen compounds fortheir effect on amino acid transport activity. By selection of hostcells that do not ordinarily express a particular amino acidtransporter, pure preparations of membranes containing the transportercan be obtained.

[0072] The recombinant expression constructs of the present inventionare useful in molecular biology to transform cells which do notordinarily express a particular amino acid transporter to thereafterexpress this receptor. Such cells are useful as intermediates for makingcell membrane preparations useful for transporter activity assays, whichare in turn useful for drug screening. The recombinant expressionconstructs of the present invention may also be useful in gene therapy.Cloned genes of the present invention, or fragments thereof, may also beused in gene therapy carried out homologous recombination orsite-directed mutagenesis. See generally Thomas & Capecchi, 1987, Cells51: 503-512; Bertling, 1987, Bioscience Reports 7: 107-112; Smithies etal., 1985, Nature 312: 230-234.

[0073] Oligonucleotides of the present invention are useful asdiagnostic tools for probing amino acid transporter gene expression intissues of humans and other animals. For example, tissues are probed insitu with oligonucleotide probes carrying detectable groups byconventional autoradiographic techniques, to investigate nativeexpression of this receptor or pathological conditions relating thereto.Further, chromosomes can be probed to investigate the presence orabsence of the corresponding amino acid transporter gene, and potentialpathological conditions related thereto.

[0074] The invention also provides antibodies that are immunologicallyreactive to the amino acid transporter protein or epitopes thereofprovided by the invention. The antibodies provided by the invention maybe raised, using methods well known in the art, in animals byinoculation with cells that express an amino acid transporter orepitopes thereof, cell membranes from such cells, whether crude membranepreparations or membranes purified using methods well known in the art,or purified preparations of proteins, including fusion proteins,particularly fusion proteins comprising epitopes of the amino acidtransporter protein of the invention fused to heterologous proteins andexpressed using genetic engineering means in bacterial, yeast oreukaryotic cells, said proteins being isolated from such cells tovarying degrees of homogeneity using conventional biochemical means.Synthetic peptides made using established synthetic means in vitro andoptionally conjugated with heterologous sequences of amino acids, arealso encompassed in these methods to produce the antibodies of theinvention. Animals that are used for such inoculations includeindividuals from species comprising cows, sheep, pigs, mice, rats,rabbits, hamsters, goats and primates. Preferred animals for inoculationare rodents (including mice, rats, hamsters) and rabbits. The mostpreferred animal is the mouse.

[0075] Cells that can be used for such inoculations, or for any of theother means used in the invention, include any cell line which naturallyexpresses the amino acid transporter provided by the invention, or anycell or cell line that expresses the amino acid transporter of theinvention, or any epitope thereof, as a result of molecular or geneticengineering, or that has been treated to increase the expression of anendogenous or heterologous amino acid transporter protein by physical,biochemical-or genetic means. Preferred cells are E. coli and insect SF9cells, most preferably E. coli cells, that have been transformed with arecombinant expression construct of the invention encoding an amino acidtransporter protein, and that express the transporter therefrom.

[0076] The present invention also provides monoclonal antibodies thatare immunologically reactive with an epitope derived from an amino acidtransporter of the invention, or fragment thereof, present on thesurface of such cells, preferably E. coli cells. Such antibodies aremade using methods and techniques well known to those of skill in theart. Monoclonal antibodies provided by the present invention areproduced by hybridoma cell lines, that are also provided by theinvention and that are made by methods well known in the art.

[0077] Hybridoma cell lines are made by fusing individual cells of amyeloma cell line with spleen cells derived from animals immunized withcells expressing an amino acid tansporter of the invention, as describedabove. The myeloma cell lines used in the invention include linesderived from myelomas of mice, rats, hamsters, primates and humans.Preferred myeloma cell lines are from mouse, and the most preferredmouse myeloma cell line is P3X63-Ag8.653. The animals from whom spleensare obtained after immunization are rats, mice and hamsters, preferablymice, most preferably Balb/c mice. Spleen cells and myeloma cells arefused using a number of methods well known in the art, including but notlimited to incubation with inactivated Sendai virus and incubation inthe presence of polyethylene glycol (PEG). The most preferred method forcell fusion is incubation in the presence of a solution of 45% (w/v)PEG-1450. Monoclonal antibodies produced by hybridoma cell lines can beharvested from cell culture supernatant fluids from in vitro cellgrowth; alternatively, hybridoma cells can be injected subcutaneouslyand/or into the peritoneal cavity of an animal, most preferably a mouse,and the monoclonal antibodies obtained from blood and/or ascites fluid.

[0078] Monoclonal antibodies provided by the present invention are alsoproduced by recombinant genetic methods well known to those of skill inthe art, and the present invention encompasses antibodies made by suchmethods that are immunologically reactive with an epitope of an aminoacid transporter of the invention. The present invention alsoencompasses fragments, including but not limited to F(ab) and F(ab)′₂fragments, of such antibody. Fragments are produced by any number ofmethods, including but not limited to proteolytic cleavage, chemicalsynthesis or preparation of such fragments by means of geneticengineering technology. The present invention also encompassessingle-chain antibodies that are immunologically reactive with anepitope of an amino acid transporter, made by methods known to those ofskill in the art.

[0079] The present invention also encompasses an epitope of an aminoacid transporter of the invention, comprised of sequences and/or aconformation of sequences present in the transporter molecule. Thisepitope may be naturally occurring, or may be the result of proteolyticcleavage of a transporter molecule and isolation of anepitope-containing peptide or may be obtained by synthesis of anepitope-containing peptide using methods well known to those skilled inthe art. The present invention also encompasses epitope peptidesproduced as a result of genetic engineering technology and synthesizedby genetically engineered prokaryotic or eukaryotic cells.

[0080] The invention also includes chimeric antibodies, comprised oflight chain and heavy chain peptides immunologically reactive to anamino acid transporter-derived epitope. The chimeric antibodies embodiedin the present invention include those that are derived from naturallyoccurring antibodies as well as chimeric antibodies made by means ofgenetic engineering technology well known to those of skill in the art.

[0081] The invention also provides methods for screening compounds fortheir ability to inhibit, facilitate or modulate the biochemicalactivity of the amino acid transporter molecules of the invention, foruse in the in vitro screening of novel agonist and antagonist compounds.In preferred embodiments, cells, particularly amphibian oocytestransformed with a recombinant expression construct of the invention arecontacted with such a compound, and the effect of the compound on thetransport of the appropriate amino acid is assayed. Additional preferredembodiments comprise quantitative analyses of such effects. Alsoprovided are assays that distinguish between the effect of suchcompounds on amino acid transport from effects of such compounds onchloride ion transport by the transporters of the invention.

[0082] The present invention is also useful for the detection ofinhibitors, analogues, agonists or antagonists, heretofore known orunknown, of the amino acid transporters of the invention, eithernaturally occurring or embodied as a drug. In preferred embodiments,such inhibitors, analogues, agonists or antagonists may be detected inblood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any otherbodily fluid. In additional preferred embodiments, the inventionprovides methods for detecting and identifying inhibitors, analogues,agonists or antagonists that preferentially affect either the amino aciduptake function or the chloride ion channel function of the amino acidtransporters of the invention.

[0083] The Examples which follow are illustrative of specificembodiments of the invention, and various uses thereof. They set forthfor explanatory purposes only, and are not to be taken as limiting theinvention.

EXAMPLE 1 Isolation of a Human Excitatory Amino Acid Transporter cDNA

[0084] Excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 havebeen disclosed in co-owned and co-pending U.S. Ser. No. 08/140,729,filed Oct. 20, 1993, now U.S. Pat. No. ______ , issued ______ , 1996,which is incorporated by reference herein in its entirety.

[0085] In order to clone a novel human excitatory amino acidtransporter, cDNA was prepared from human cerebellar mRNA and amplifiedin vitro using a pair of degenerate primers derived from two regions ofsequence similarity between the EAAT1, EAAT2 and EAAT3 genes previouslydisclosed. Briefly, total RNA was isolated using the method ofChomczynski & Sacchi (1987, Anal. Biochem. 162 : 156-159), wherein thetissue is disrupted and solubilized in a solution containing guanidiniumisothiocyanate and the RNA purified by phenol/chloroform extractions.Total cellular RNA thus isolated was then enriched for poly (A⁺) mRNA byoligo (dT) chromatography. A mixture of oligo (dT)-primed andrandom-primed mRNA was converted to cDNA using the Superscript ChoiceSystem (Bethesda Research Labs, Gaithersburg, Md.). 10 ng of thiscerebellum-derived cDNA preparation was then amplified using thefollowing degenerate oligonucleotide primers: Sense primer:5′-CGCGGGTACCAA(T/C)CT(C/G)GT(C/A/G)(G/C)A(G/A)GC(T/C)TG(T/C)TT(T/C)-3′;(SEQ ID NO:9) Antisense primer:5′-CGCGTCTAGA(T/C)TG(A/G/T)GC(A/G/T)AT(A/G)AA(A/G)A(T/C)(G/T/C)GC(A/G/T)GC-3′.(SEQ ID NO:10)

[0086] PCR amplification was performed for 35 cycles, each cyclecomprising 1 minute at 94° C., 1 min at 47° C. and 2 minutes at 72° C.using Vent polymerase (New England Biolabs, Beverly, Mass.). Followingthe PCR, the product of the amplification reaction was purified usingstandard techniques (Saiki et al., 1988, Science 239: 487-491).

[0087] Novel candidate clones were identified by the followinghybridization strategy. Bona fide transporter cDNAs were identified byhybridization with the following degenerate oligonucleotide probe for ahighly conserved coding sequence:

[0088]5′-CTGRGCRATGAARATGGCAGCCAGGGCYTCATACAGGGCTGTGCCRTCCATGTTRATGGTRGC-3′(SEQ ID No.:11)

[0089] (See Arriza et al., 1992, ibid.). Those transporter cDNAspreviously isolated (i.e., encoding EAAT1, EAAT2, EAAT3, ASCT1) wereidentified by specific hybridization with their previously-isolated cDNAprobes under high stringency conditions (e.g., 0. 1×SSC (standardcitrate saline)/1%SDS (sodium dodecyl sulfate) at 65° C. (see Sambrooket al., 1990, Molecular Cloning: A Laboratory Manual (Cold Spring HarborPress: New York))). cDNAs that hybridized with the oligonucleotide butdid not correspond to previously isolated cDNAs were characterized bynucleotide sequence analysis.

[0090] A novel clone similar to the previously-identified glutamatetransporters was identified. This novel PCR amplification product wasused to screen a motor cortex mRNA prepared using standard techniques(see Sambrook et al., ibid.). Total RNA was isolated using the method ofChomczynski & Sacchi as above, and then enriched for poly (A⁺) mRNA byoligo (dT) chromatography. A mixture of oligo (dT)-primed andrandom-primed mRNA was converted to cDNA using the Superscript ChoiceSystem and the cDNA was then ligated into the cloning vector λZAPII(Strategene, La Jolla, Calif.), packaged into phage heads usingcommercially-available packaging extracts (Strategene) and used toinfect E. coli. Lawns of infected bacterial cells were used to makeplaque lifts for hybridization using standard conditions (see Sambrook,et al., ibid.).

[0091] This cDNA library was hybridized with the ³²P-labeled PCRamplification product described above. Hybridization was performed at50° C. in a solution containing 0.5M Na₂HPO₄ (pH 7.15)/7% sodium dodecylsulfate (SDS) and the filters were washed at 60° C. in 2×SSPE (0.36MNaCl/20 mM sodium phosphate (pH 7.7)/2 mM ethylenediamine tetraaceticacid (EDTA)) and 1% SDS. Hybridizing clones were identified byautoradiography at −70° C. using tungsten-containing intensifyingscreens (DuPont-NEN, Wilmington, Del.).

[0092] Two independent clones containing 2.2 and 2.3 kilobase pair (kb)cDNA inserts were isolated. cDNA inserts were excised from the cloningvector in vivo by superinfection with a defective filamentous phage thatrecognizes and excises cloned insert sequences along with adjacentmodified phage replication-form sequences (termed pBluescript SK andavailable from Strategene). Each clone was then sequenced using thedideoxy-chain termination method of Sanger et al. (1977, Proc. Natl.Acad. Sci. USA 74: 5463), using Sequenase 2.0, a modified form ofbacteriophage T7 DNA polymerase (U.S. Biochemical Corp., Cleveland,Ohio). The two independent clones contained identical long open readingframes, and were identical in those regions of the 3′ untranslatedsequences analyzed. Each clone had a distinct 5′ untranslated sequencebut were identical in the 8 nucleotides upstream of the putativetranslation start site. This observation may be indicative ofalternative RNA splicing in the 5′ untranslated region of the EAAT4gene. The 1734 nucleotide sequence of the human EAAT4 cDNA shown in FIG.1 includes the conserved 8 nucleotides of the 5′ untranslated region, 34nucleotides of the 3′ untranslated region downstream of the translationstop codon, and 1692 nucleotides of coding sequence comprising 564 aminoacids (FIG. 2 and FIG. 3).

[0093] The EAAT4 amino acid sequence (SEQ ID No.:2; shown in FIG. 2) wasfound to exhibit similarity to other known glutamate transportersubtypes (an amino acid sequence comparison is shown in FIG. 3). Anamino acid comparison between these 4 human glutamate transportersshowed 65%, 41% and 48% sequence identity (respectively) between EAAT4and EAAT1, EAAT2 and EAAT3 (shown in FIG. 3 by shaded boxes). Both theamino and carboxyl termini were found to be divergent between thesetransporter proteins, and diversity was also found in the extracellulardomains of these putative protein sequences, which contain conservedpotential N-glycosylation sites (shown in FIG. 3 by open boxes). It wasnoted that previously-identified sequence (comprising the amino acids--AA(I/V)FIAQ--) that was highly conserved in the glutamate transporterswas also present in EAAT4 (at positions 434-440 of the EAAT4 amino acidsequence shown in FIG. 2). 6-10 putative transmembrane domains werefound using the algorithm of Eisenberg et al. (1984, J. Molec. Biol.179: 125-142). On the basis of these data EAAT4 was determined to encodea related but distinct and novel member of the excitatory amino acidtransporter family.

EXAMPLE 2 Tissue Distribution of Amino Acid Transporter Expression

[0094] The tissue distribution of mRNA corresponding to expression ofthe EAAT4 amino acid transporter disclosed herein was determined invarious tissues by Northern hybridization experiments (see Sambrook etal., ibid.). The results of these experiments are shown in FIGS. 4 and5.

[0095] A panel of tissue samples was examined by Northern hybridizationanalysis performed under high stringency conditions as follows. A nylonfilter containing 2 μg human peripheral tissue poly(A)⁺ RNA was obtainedfrom Clonetech Laboratories (Palo Alto, Calif.), and a similar filterwas prepared containing human brain region RNA as follows. Total RNA wasisolated from human brain region tissue obtained from the Oregon BrainRepository and 20 μg/ region were size-fractionated by denaturingformaldehyde agarose gel electrophoresis (see Sambrook et al., ibid.).Fractionated RNA was then transferred to a nylon filter using theNorthern blot/capillary-osmotic technique. Northern hybridization ofboth filters was performed individually with a ³²P-labeled 1.7 kbEAAT4-specific probe prepared by random-primed synthesis (BoehringerMannheim, Indianapolis, Ind.) using α-³²P-labeled dCTP. Filters werehybridized overnight at 42° C. individually with each radiolabeled probe(at a concentration of 10⁶ cpm/mL) in a solution of 5×/SSPE/50%formamide/7.5×Denhardt's solution (comprising 0.15g/100 mL each ofFicoll, polyvinylpyrrolidone and bovine serum albumin)/2% SDS and 100μg/mL denatured salmon-sperm DNA. Following hybridization, filters werewashed twice for 30 min at room temperature in 2×SSPE/0.1% SDS and twicefor 20 min at 50° C. in 0.1×SSPE/0. 1% SDS. Hybridizing RNAs werevisualized by autoradiography at −70° C. using intensifying screens. Thefilters were subsequently re-probed as described with a radiolabeledhuman β-actin probe (Clonetech) as a positive control.

[0096] The results of these experiments, shown in FIGS. 4 and 5,demonstrate that EAAT4 transporter has a distribution distinct fromother transporters isolated to date. FIG. 4 shows the distribution ofthese amino acid transporter transcripts in different human brainregions. Expression of the 2.4 kb EAAT4 mRNA was detected only incerebellum using this Northern blot assay. However, a more sensitivepolymerase chain reaction-based assay revealed EAAT4 expression in brainstem, cortex and hippocampus, albeit at much lower levels than thoseseen in cerebellum.

[0097]FIG. 5 illustrates expression of the EAAT4 transporter in humanheart, brain, placenta, lung, liver, muscle, kidney and pancreas. Thesize (in kb) of the transcripts corresponding to expression of thistransporter are displayed along the right-hand border of each panel. Asis seen from these autoradiographs, EAAT4 was found to be expressedpredominantly in brain and placenta as a single, 2.4 kb transcript,consistent with the size of the isolated cDNA clones.

[0098] These results support the conclusion that the amino acidtransporter of the invention may play an important role in normal brainfunction, and that disruption of amino acid transport by thistransporter may be important determinants in organic brain dysfunction,as a result of ischemia or anoxia. Moreover, because of the presumedrole of the cerebellum in motor learning, the abundant expression ofEAAT4 mRNA in the cerebellum suggests that this protein may besignificant in both normal and dysfunctional (e.g., ataxia and relatedsyndromes) motor coordination.

EXAMPLE 3 Functional Expression of the EAAT4 Amino Acid Transporter Genein Xenopus Oocytes

[0099] The sequence similarity between EAAT4 and thepreviously-identified glutamate transporters EAAT1, EAAT2 and EAAT3suggested that the protein encoded by the EAAT4 cDNA was an amino acidtransporter. The ability of the EAAT4 gene product to transport aminoacids, and the identity of which amino acids might be transported bythis gene product, was assayed in Xenopus oocytes followingmicroinjection of in vitro synthesized EAAT4 RNA.

[0100] Briefly, the coding sequence of the EAAT4 cDNA was isolated withunique flanking restriction sites using a PCR-based assay. In thisassay, each of the complementary primers used for PCR amplification ofthe coding sequence contained a sequence encoding a unique restrictionenzyme recognition site at the 5′ terminus of each PCR primer. ForEAAT4, the sense primer contained a HindIII recognition sequence(A↓AGCTT), and the antisense primer contained an XbaI recognitionsequence (T↓CTAGA) at their respective 5′ termini. Each of the PCRprimers used for amplifying EAAT4 sequences had the following sequence:EAAT4 sense primer: (SEQ ID NO:12)5′-GCGCGTCGACAAGCTTGCCATGCAACAGCCTGTT-3′; EAAT4 antisense primer: (SEQID NO:13) 5′-GCGCTCTAGATCAGCCCACGGTCAGTTG-3′.

[0101] PCR amplification was performed for 30 cycles, each cyclecomprising 30 seconds at 94° C., 30 seconds at 55° C. and 1 minute at72° C. Following the PCR, the product of the amplification reaction waspurified using standard techniques (Saiki et al., 1988, Sciences:487-491). The DNA then digested with the restriction enzymes HindIII andXbaI and then cloned into the polylinker of an oocyte transcriptionvector (pOTV; see Arriza et al., 1992, ibid.). Capped RNA wassynthesized from linearized plasmid employing bacteriophage T7 RNApolymerase (mMessage mMachine; Ambion, Austin, Tex.), diluted with waterto a concentration of 400 μg/mL, and 50 nL of this EAAT4 RNA wasinjected into defolliculated stage V or VI oocytes. Oocytes wereprepared as described (Quick and Lester, 1994, Methods in Neuroscience19: 261-279) and maintained at 17° C. for up to 5 days.

[0102] Amino acid transport in these oocytes was assayed at roomtemperature 3-5 days post injection using various concentrations of[³H]-L-aspartate or [³H]-L-glutamate (obtained from New England Nuclear,Boston, Mass.) in ND96 buffer (96 mM NaCl/2 mM KCl/1.8 mM CaCl₂/1 mMMgCl₂/5 mM Hepes, pH 7.5).

[0103] Two electrode voltage clamp recordings from EAAT4expressingoocytes were performed at room temperature using glass microelectrodesfilled with 3M KCl solutions (resistance <1 MΩ) and a Ag/AgCl pelletbath ground or an active bath probe. An Axon GeneClamp 500 amplifier wasused with Digidata 1200 interfaces. The pClamp suite of programs (AxonInstruments, Foster City, Calif.) was used to control stimulationparameters, for data acquisition and analysis. MacLab data acquisitionsoftware and a MacLab/2e interface (ADI Instruments, Milford, Mass.)were used to record electrophysiological experiments. 3M KCl/agarbridges were used to avoid offset voltages associated with bufferchanges. Oocytes were continuously superfused with ND96 or ionsubstituted ND96 during the recording session.

[0104] The properties of EAAT4 cRNA-injected oocytes were examined byanalysis of excitatory amino acid-induced currents in voltage-clampedoocytes. In these experiments, the maximum current of depolarization(I_(max)) and the transport constant (K_(m)) induced by treatment with avariety of putative EAAT4 transporter substrates was determined. Theresults of these experiments are shown in Table I. Injected oocytes werealso incubated with a mixture of radiolabeled amino acid([³H]-L-glutamate (17.8 Ci/mmol) or [³H]-D-aspartate (15.5 Ci/mmol);Dupont-NEN) and nonradiolabeled amino acid at increasing doses of 0.1,0.3, 1, 3, 10 and 100 μM for 10 min. for determination of the V_(max),and K_(m), of substrate flux. After incubation, the cells were washedfour times with ice-cold PBS, solubilized with a solution of 0.1% sodiumdodecyl sulfate (SDS) and the amount of radioactivity associated withthe cells determined using standard liquid scintillation countingmethods.

[0105] Application of L-aspartate or L-glutamate to EAAT4 cRNA-injected,voltage-clamped oocytes induced dose-dependent, saturable inwardcurrents; no such currents were observed in control, mock-injectedoocytes. Flux experiments showed that oocytes injected with the EAAT4excitatory amino acid transporter-encoding cRNA accumulatedsignificantly-higher (about 30-fold higher) saturable, sodium-dependentuptake of [³H]-L-aspartate and [³H]-L-glutamate than did mock-injectedoocytes. The maximal currents elicited by other test compounds werenormalized relative to the maximal current obtained with a saturatingdose (100 μM) of L-aspartate in the same oocyte such that thesedeterminations were independent of the level of EAAT4 expression inindividual oocytes. For this normalization, the current elicited by amaximal dose of L-aspartate was measured before and/or after doseresponse determinations for each compound and given a normalized I_(max)value of 1.0. No compound tested induced a depolarizing current inuninjected oocytes or water-injected oocytes. The course of uptake of 10μM radioactive aspartate was found to be linear for at least 20 min inassays performed at room temperature. The K_(m) values derived from thecurrent measurements as well as radiolabel substrate uptake experiments,as shown in Table I, were about 10-fold lower than those found forpreviously-disclosed members of the EAAT transporter family. Maximalflux and current measurements (Table 1) indicated a discrepancy betweenthe maximal transport and maximal current generated by L-aspartate andL-glutamate. Although L-aspartate produced a current significantlylarger than L-glutamate (1.0 versus 0.37, respectively), its maximaltransport rate is significantly lower than that of L-glutamate (3.26versus 5.72). This data suggested that the substrate elicited currentsobserved are not a direct reflection of substrate transport and that thetwo phenomena may be uncoupled.

[0106] Structural analogues of glutamate, includingL-trans-2,4-pyrrolidine dicarboxylic acid, L-quisqualate and L-α-aminoadipate, also elicited currents of varying magnitude in EAAT4-injectedoocytes, while kainic acid, which blocks glutamate transport in somebrain regions, was found to be ineffective in either eliciting a currentor blocking an L-aspartate induced current at concentrations as high as5 mM. These properties of the EAAT4 transporter are consistent withglutamate uptake activity previously reported for cerebellum and areconsistent with localization of EAAT4 expression primarily in cerebellartissues.

[0107] Further characterization of the electrophysiology of EAAT4accompanying aspartate transport revealed a significant differencebetween EAAT4 and the previously-disclosed members of the EAATtransporter family. L-glutamate- and L-aspartate-induced currents foundin voltage-clamped oocytes expressing these other members of the EAATfamily exhibited strong inward rectification (i.e., currents wereinduced that were preferentially inward) and were not found to reversebelow +40 mV. In contrast, 10 μM L-aspartate was found to induce anoutward current in EAAT4-injected oocytes at voltages more positive than˜−20 mV. Results of experiments demonstrating this phenomenon are shownin FIG. 6A. In FIG. 6B it is shown that the voltage-dependence of theL-aspartate induced current is approximately linear from about −100 mVto about +40 mV, reversing at −22±1.6 mV (n=18). This value isapproximately equal to the previously-reported equilibrium potential ofchloride ion in native Xenopus oocytes (−24 mV; see Barish, 1983, J.Physiol. (London) 342: 309-325).

[0108] To investigate the significance of this coincidence, the chlorideion-dependence was assayed by substituting chloride ion with gluconateion. The reversal potential of the L-aspartate induced currents werefound to shift by 57±1.5 mV for every ten-fold change in chloride ionconcentration, as shown in FIGS. 6B and 6C. This result is in accordwith theoretical predictions of the Nernst equation for chloride-ionselective conductance. Similar results were obtained using L-glutamate(reversal potential shifted 50±2 mV for every ten-fold change inchloride ion concentration). The difference in the reversal potentialshift seen with L-glutamate and L-aspartate (50 mV versus 57 mV) resultsfrom differences in their relative efficacies as transport substrates(Glu>Asp) and as elicitors of the chloride conductance (Asp>Glu).

[0109] Complete removal of chloride by substitution with gluconate ionabolished the L-aspartate induced outward currents at depolarizedpotentials (see FIG. 6B). However, L-aspartate transport at −6 mV in theabsence of chloride ion (V_(max)=4.7±0.3 fmol/s, n=8) was notsignificantly different than transport in the presence of chloride ion(V_(max)=5.9 ±0.3 fmol/s, n=8), indicating that transport is notdependent on external chloride ion. In contrast, substitution of sodiumions with choline completely and reversibly abolishedL-aspartate-induced currents, consistent with the sodium dependence ofuptake and with the sodium-dependent activation of chloride conductanceby excitatory amino acids (FIG. 6D). TABLE I Pharmacological Propertiesof EAAT4 Substrate Flux and Conductance Currents Amino Acid K_(m) (μM)V_(max) [³H]-L-aspartate 0.97 ± 0.29 3.26 ± 0.06 [³H]-L-glutamate 2.49 ±0.87 5.72 ± 0.52 Amino Acid K_(m) (μM) I_(max) L-aspartate 1.84 ± 0.461.0 L-glutamate 3.3 ± 0.4 0.37 ± 0.07 D-aspartate 2.5 ± 0.3 1.0 ± 0.1L-α-adipate 168 ± 11  0.75 ± 0.02 L-quisqualate 99 ± 11 0.47 ± 0.06L-homocysteate 403 ± 72  0.83 ± 0.05 PDC* 2.6 ± 0.4 0.41 ± 0.01 Kainicacid >5000 undetectable

[0110] The magnitude of the induced chloride conductance in EAAT4distinguishes it from EAAT1, EAAT2, or EAAT3. EAAT1 through EAAT3 havelarge transport currents with small or undetectable chlorideconductances, such that their current/voltage profiles show inwardrectification, i.e., at positive potentials the current is reduced butis not reversed or outward at potentials less than +40 mV. This isillustrated by the I-V profile for EAAT2 shown in FIG. 7A. In contrast,the EAAT4 I-V profile does not show rectification; rather, the profileis linear with the reversal potential ˜−20 mV, and the reversalpotential is constant regardless of the dose of L-aspartate applied(FIG. 7B). Flux studies under voltage-clamp conditions were conductedand fit to the Faraday equation. It was concluded that greater than 95%of the L-aspartate induced current in EAAT4-expressing oocytes can beattributed to chloride conductance.

[0111]L-aspartate and L-glutamate were found to differ in the degree towhich they elicit either the transport current component or the chlorideconductance component of EAAT4 (FIG. 7C). This is reflected by the factthat, although the reversal potential for L-aspartate was ˜−20 mV,L-glutamate elicited a more robust transport component and therefore itsmeasured reversal potential was ˜−3 mV. Various compounds were testedand a characteristic rank order for reversal potential (more negative toless negative) was found:L-aspartate>PDC>L-α-aminoadipate>L-glutamate>L-quisqualate.

[0112] These data demonstrated that the EAAT4 transporter is capable ofmediating both 1) the uptake of excitatory amino acids such as glutamateand aspartate, and 2) a substrate-gated increase in chlorideconductance. Various substrates can be more or less effective as eitheramino acid uptake inhibitors or as elicitors of chloride conductance.Thus, the pharmacology of the two functions are separable and thereforesubject to selective interventions, i.e., compounds that selectivelyinhibit or stimulate chloride ion conductance without affectingexcitatory amino acid uptake.

[0113] The physiological significance of the large chloride conductanceof EAAT4 is unknown; but like the chloride conductance gated by GABA-Areceptors, it may have a general inhibitory affect on neurotransmission.It may therefore be therapeutically beneficial to selectively stimulateor inhibit the EAAT4 chloride conductance in diseases such as epilepsy,excitotoxicity, and neurodegenerative diseases. The separability of thetwo biochemical functions of amino acid uptake and chloride ionconductance in the EAAT4 provides an assay for detecting and identifyingcompounds that inhibit or stimulate the chloride ion conductance of theEAAT4 transporter but do not affect amino acid uptake. Such compoundsare advantageous, due to the recognized hazards of pharmaceutical agentsthat inhibit amino acid uptake in the brain. The assays described hereinprovide a method for screening for agents that can selectively inhibitor stimulate chloride conductance without the risk of brain injuryassociated with concommitant inhibition of amino acid uptake.

[0114] Briefly, such assays are performed as follows, usingvoltage-clamped oocytes expressing EAAT4 as described above. First, thecompounds are tested for the ability to elicit a current in thevoltage-clamped, EAAT4-expressing oocytes. If the compound does notelicit a current, the compound is then co-applied to the oocytes in thepresence of L-aspartate to determine whether the compound is aninhibitor of the aspartate-elicited current. If the tested compoundelicits a current, the relationship between the elicited current and thevoltage is determined (as shown in FIGS. 6 and 7). This analysis yieldsthe relative current amplitude, the reversal potential and the degree ofrectification caused by treatment of the oocyte with the test compound.As described above, the reversal potential and degree of rectificationreflect the relative contributions of the uptake-elicited current andthe chloride conductance in the response of the oocyte to the testcompound.

[0115] Compounds which elicit a current which is determined to have asignificant chloride conductance component are then tested for theireffect on excitatory amino acid uptake. Mammalian cells or oocytes aretreated with detectably-labeled L-aspartate and L-glutamate in thepresence or absence of a compound to be tested. Compounds having littleeffect on excitatory amino acid uptake, particularly L-glutamate uptake,are thereby advantageously distinguished.

EXAMPLE 4 Construction of Fusion Proteins-Recombinant ExpressionConstructs for Expression of Immunologically-Active Epitopes of AminoAcid Transporters

[0116] The EAAT4 amino acid transporter protein of the invention areexpressed as fusion proteins in bacteria to produceimmunologically-active epitopes. In these experiments, the amino acidtransporter cDNAs of the invention are excised from their respectivepOTV-containing constructs and subcloned into a pGEX-2T construct(Pharmacia, Piscataway, N.J.) whereby the coding sequences of the aminoacid transporter cDNA is translationally in-frame with sequencesencoding glutathione-S-transferase (described in Arriza et al., 1992, J.Neurosci. 12: 4045-4055), termed pGST-EAAT4-constructs. Afterintroduction of the pGST-EAAT4 constructs into bacterial cells (E. coli,strain D5α) using conventional techniques (see Sambrook et al., ibid.),fusion protein expression is induced withisopropyl-1-thio-β-D-galactopyranoside as described (Smith & Johnson,1988, Gene 67: 31-40) and purified using glutathione-Sepharose 4B(Pharmacia). Antibodies are then raised against the amino acidtransporter of the invention by inoculation of rabbits with 300-500 μgof purified fusion protein in Freund's adjuvant (Grand Island BiologicalCo., Grand Island, N.Y.), said inoculation repeated approximately every4 weeks. Sera are immunoaffinity-purified on columns of Affi-Gel 15derivatized with purified fusion protein. After salt elution, suchantibodies are neutralized, stabilized with bovine serum albumin at afinal concentration of 1 mg/mL, dialyzed against PBS and assayed byimmunoblotting using conventional techniques (Harlow & Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

[0117] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

1 13 1734 base pairs nucleic acid single linear cDNA CDS 9..1703 5′UTR1..8 3′UTR 1704..1734 1 GATAGACC ATG AGC AGC CAT GGC AAC AGC CTG TTC CTTCGG GAG AGC GGC 50 Met Ser Ser His Gly Asn Ser Leu Phe Leu Arg Glu SerGly 1 5 10 CAG CGG CTG GGC CGG GTG GGC TGG CTG CAG CGG CTG CAG GAA AGCCTG 98 Gln Arg Leu Gly Arg Val Gly Trp Leu Gln Arg Leu Gln Glu Ser Leu15 20 25 30 CAG CAG AGA GCA CTG CGC ACG CGC CTG CGC CTG CAG ACC ATG ACCCTC 146 Gln Gln Arg Ala Leu Arg Thr Arg Leu Arg Leu Gln Thr Met Thr Leu35 40 45 GAG CAC GTG CTG CGC TTC CTG CGC CGA AAC GCC TTC ATT CTG CTG ACG194 Glu His Val Leu Arg Phe Leu Arg Arg Asn Ala Phe Ile Leu Leu Thr 5055 60 GTC AGC GCC GTG GTC ATT GGG GTC AGC CTG GCC TTT GCC CTG CGC CCA242 Val Ser Ala Val Val Ile Gly Val Ser Leu Ala Phe Ala Leu Arg Pro 6570 75 TAT CAG CTC ACC TAC CGC CAG ATC AAG TAC TTC TCT TTT CCT GGA GAG290 Tyr Gln Leu Thr Tyr Arg Gln Ile Lys Tyr Phe Ser Phe Pro Gly Glu 8085 90 CTT CTG ATG AGG ATG CTG CAG ATG CTG GTG TTA CCT CTC ATT GTC TCC338 Leu Leu Met Arg Met Leu Gln Met Leu Val Leu Pro Leu Ile Val Ser 95100 105 110 AGC CTG GTC ACA GGT ATG GCA TCC CTG GAC AAC AAG GCC ACG GGGCGG 386 Ser Leu Val Thr Gly Met Ala Ser Leu Asp Asn Lys Ala Thr Gly Arg115 120 125 ATG GGG ATG CGG GCA GCT GTG TAC TAC ATG GTG ACC ACC ATC ATCGCG 434 Met Gly Met Arg Ala Ala Val Tyr Tyr Met Val Thr Thr Ile Ile Ala130 135 140 GTC TTC ATC GGC ATC CTC ATG GTC ACC ATC ATC CAT CCC GGG AAGGGC 482 Val Phe Ile Gly Ile Leu Met Val Thr Ile Ile His Pro Gly Lys Gly145 150 155 TCC AAG GAG GGG CTG CAC CGG GAG GGC CGG ATC GAG ACC ATC CCCACA 530 Ser Lys Glu Gly Leu His Arg Glu Gly Arg Ile Glu Thr Ile Pro Thr160 165 170 GCT GAT GCC TTC ATG GAC CTG ATC AGA AAT ATG TTT CCA CCA AACCTT 578 Ala Asp Ala Phe Met Asp Leu Ile Arg Asn Met Phe Pro Pro Asn Leu175 180 185 190 GTG GAG GCC TGC TTC AAA CAG TTC AAG ACG CAG TAC AGC ACGAGG GTG 626 Val Glu Ala Cys Phe Lys Gln Phe Lys Thr Gln Tyr Ser Thr ArgVal 195 200 205 GTA ACC AGG ACC ATG GTG AGG ACA GAG AAC GGG TCT GAG CCGGGT GCC 674 Val Thr Arg Thr Met Val Arg Thr Glu Asn Gly Ser Glu Pro GlyAla 210 215 220 TCC ATG CCT CCT CCA TTC TCA GTG GAG AAC GGA ACC AGC TTCCTG GAA 722 Ser Met Pro Pro Pro Phe Ser Val Glu Asn Gly Thr Ser Phe LeuGlu 225 230 235 AAT GTC ACT CGG GCC TTG GGT ACC CTG CAG GAG ATG CTG AGCTTT GAG 770 Asn Val Thr Arg Ala Leu Gly Thr Leu Gln Glu Met Leu Ser PheGlu 240 245 250 GAG ACT GTA CCC GTG CCT GGC TCC GCC AAT GGC ATC AAC GCCCTG GGC 818 Glu Thr Val Pro Val Pro Gly Ser Ala Asn Gly Ile Asn Ala LeuGly 255 260 265 270 CTC GTG GTC TTC TCT GTG GCC TTT GGG CTG GTC ATT GGTGGC ATG AAA 866 Leu Val Val Phe Ser Val Ala Phe Gly Leu Val Ile Gly GlyMet Lys 275 280 285 CAC AAG GGC AGA GTC CTC AGG GAC TTC TTC GAC AGC CTCAAT GAG GCT 914 His Lys Gly Arg Val Leu Arg Asp Phe Phe Asp Ser Leu AsnGlu Ala 290 295 300 ATT ATG AGG CTG GTG GGC ATC ATT ATC TGG TAT GCA CCTGTG GGC ATC 962 Ile Met Arg Leu Val Gly Ile Ile Ile Trp Tyr Ala Pro ValGly Ile 305 310 315 CTG TTC CTG ATT GCT GGG AAG ATT CTG GAG ATG GAA GACATG GCC GTC 1010 Leu Phe Leu Ile Ala Gly Lys Ile Leu Glu Met Glu Asp MetAla Val 320 325 330 CTG GGG GGT CAG CTG GGC ATG TAC ACC CTG ACC GTC ATCGTG GGC CTG 1058 Leu Gly Gly Gln Leu Gly Met Tyr Thr Leu Thr Val Ile ValGly Leu 335 340 345 350 TTC CTC CAT GCC GGC ATT GTC CTT CCC CTC ATC TACTTC CTC GTC ACT 1106 Phe Leu His Ala Gly Ile Val Leu Pro Leu Ile Tyr PheLeu Val Thr 355 360 365 CAC CGG AAC CCC TTC CCC TTC ATT GGG GGC ATG CTACAA GCC CTC ATC 1154 His Arg Asn Pro Phe Pro Phe Ile Gly Gly Met Leu GlnAla Leu Ile 370 375 380 ACC GCT ATG GGC ACG TCT TCC AGC TCG GCA ACG CTGCCC ATC ACC TTC 1202 Thr Ala Met Gly Thr Ser Ser Ser Ser Ala Thr Leu ProIle Thr Phe 385 390 395 CGC TGC CTG GAG GAG GGC CTG GGT GTG GAC CGC CGCATC ACC AGG TTC 1250 Arg Cys Leu Glu Glu Gly Leu Gly Val Asp Arg Arg IleThr Arg Phe 400 405 410 GTC CTG CCC GTG GGC GCC ACG GTC AAC ATG GAT GGCACT GCC CTC TAC 1298 Val Leu Pro Val Gly Ala Thr Val Asn Met Asp Gly ThrAla Leu Tyr 415 420 425 430 GAG GCC CTG GCT GCC ATC TTC ATT GCT CAA GTTAAC AAC TAC GAG CTC 1346 Glu Ala Leu Ala Ala Ile Phe Ile Ala Gln Val AsnAsn Tyr Glu Leu 435 440 445 AAC CTG GGT CAG ATC ACA ACC ATC AGC ATC ACGGCC ACA GCA GCC AGT 1394 Asn Leu Gly Gln Ile Thr Thr Ile Ser Ile Thr AlaThr Ala Ala Ser 450 455 460 GTT GGG GCT GCT GGC ATC CCC CAG GCG GGT CTGGTC ACC ATG GTC ATT 1442 Val Gly Ala Ala Gly Ile Pro Gln Ala Gly Leu ValThr Met Val Ile 465 470 475 GTG CTT ACG TCG GTC GGC TTG CCC ACG GAA GACATC ACG CTC ATC ATC 1490 Val Leu Thr Ser Val Gly Leu Pro Thr Glu Asp IleThr Leu Ile Ile 480 485 490 GCC GTG GAC TGG TTC CTT GAC CGG CTT CGC ACAATG ACC AAC GTA CTG 1538 Ala Val Asp Trp Phe Leu Asp Arg Leu Arg Thr MetThr Asn Val Leu 495 500 505 510 GGC CAC TCA ATT GGA GCG GCC GTC ATC GAGCAC TTG TCT CAG CGG GAG 1586 Gly His Ser Ile Gly Ala Ala Val Ile Glu HisLeu Ser Gln Arg Glu 515 520 525 CTG GAG CTT CAG GAA GCT GAG CTT ACC CTCCCC AGC CTG GGG AAA CCC 1634 Leu Glu Leu Gln Glu Ala Glu Leu Thr Leu ProSer Leu Gly Lys Pro 530 535 540 TAC AAG TCC CTC ATG GCA CAG GAG AAG GGGGCA TCC CGG GGA CGG GGA 1682 Tyr Lys Ser Leu Met Ala Gln Glu Lys Gly AlaSer Arg Gly Arg Gly 545 550 555 GGC AAC GAG AGT GCT ATG TGAGGGGCCTCCAGCTCTGC CCCCCCAGAG AGGA 1734 Gly Asn Glu Ser Ala Met 560 565 564amino acids amino acid linear protein 2 Met Ser Ser His Gly Asn Ser LeuPhe Leu Arg Glu Ser Gly Gln Arg 1 5 10 15 Leu Gly Arg Val Gly Trp LeuGln Arg Leu Gln Glu Ser Leu Gln Gln 20 25 30 Arg Ala Leu Arg Thr Arg LeuArg Leu Gln Thr Met Thr Leu Glu His 35 40 45 Val Leu Arg Phe Leu Arg ArgAsn Ala Phe Ile Leu Leu Thr Val Ser 50 55 60 Ala Val Val Ile Gly Val SerLeu Ala Phe Ala Leu Arg Pro Tyr Gln 65 70 75 80 Leu Thr Tyr Arg Gln IleLys Tyr Phe Ser Phe Pro Gly Glu Leu Leu 85 90 95 Met Arg Met Leu Gln MetLeu Val Leu Pro Leu Ile Val Ser Ser Leu 100 105 110 Val Thr Gly Met AlaSer Leu Asp Asn Lys Ala Thr Gly Arg Met Gly 115 120 125 Met Arg Ala AlaVal Tyr Tyr Met Val Thr Thr Ile Ile Ala Val Phe 130 135 140 Ile Gly IleLeu Met Val Thr Ile Ile His Pro Gly Lys Gly Ser Lys 145 150 155 160 GluGly Leu His Arg Glu Gly Arg Ile Glu Thr Ile Pro Thr Ala Asp 165 170 175Ala Phe Met Asp Leu Ile Arg Asn Met Phe Pro Pro Asn Leu Val Glu 180 185190 Ala Cys Phe Lys Gln Phe Lys Thr Gln Tyr Ser Thr Arg Val Val Thr 195200 205 Arg Thr Met Val Arg Thr Glu Asn Gly Ser Glu Pro Gly Ala Ser Met210 215 220 Pro Pro Pro Phe Ser Val Glu Asn Gly Thr Ser Phe Leu Glu AsnVal 225 230 235 240 Thr Arg Ala Leu Gly Thr Leu Gln Glu Met Leu Ser PheGlu Glu Thr 245 250 255 Val Pro Val Pro Gly Ser Ala Asn Gly Ile Asn AlaLeu Gly Leu Val 260 265 270 Val Phe Ser Val Ala Phe Gly Leu Val Ile GlyGly Met Lys His Lys 275 280 285 Gly Arg Val Leu Arg Asp Phe Phe Asp SerLeu Asn Glu Ala Ile Met 290 295 300 Arg Leu Val Gly Ile Ile Ile Trp TyrAla Pro Val Gly Ile Leu Phe 305 310 315 320 Leu Ile Ala Gly Lys Ile LeuGlu Met Glu Asp Met Ala Val Leu Gly 325 330 335 Gly Gln Leu Gly Met TyrThr Leu Thr Val Ile Val Gly Leu Phe Leu 340 345 350 His Ala Gly Ile ValLeu Pro Leu Ile Tyr Phe Leu Val Thr His Arg 355 360 365 Asn Pro Phe ProPhe Ile Gly Gly Met Leu Gln Ala Leu Ile Thr Ala 370 375 380 Met Gly ThrSer Ser Ser Ser Ala Thr Leu Pro Ile Thr Phe Arg Cys 385 390 395 400 LeuGlu Glu Gly Leu Gly Val Asp Arg Arg Ile Thr Arg Phe Val Leu 405 410 415Pro Val Gly Ala Thr Val Asn Met Asp Gly Thr Ala Leu Tyr Glu Ala 420 425430 Leu Ala Ala Ile Phe Ile Ala Gln Val Asn Asn Tyr Glu Leu Asn Leu 435440 445 Gly Gln Ile Thr Thr Ile Ser Ile Thr Ala Thr Ala Ala Ser Val Gly450 455 460 Ala Ala Gly Ile Pro Gln Ala Gly Leu Val Thr Met Val Ile ValLeu 465 470 475 480 Thr Ser Val Gly Leu Pro Thr Glu Asp Ile Thr Leu IleIle Ala Val 485 490 495 Asp Trp Phe Leu Asp Arg Leu Arg Thr Met Thr AsnVal Leu Gly His 500 505 510 Ser Ile Gly Ala Ala Val Ile Glu His Leu SerGln Arg Glu Leu Glu 515 520 525 Leu Gln Glu Ala Glu Leu Thr Leu Pro SerLeu Gly Lys Pro Tyr Lys 530 535 540 Ser Leu Met Ala Gln Glu Lys Gly AlaSer Arg Gly Arg Gly Gly Asn 545 550 555 560 Glu Ser Ala Met 1680 basepairs nucleic acid single linear cDNA 5′UTR 1..30 CDS 31..1656 3′UTR1657..1680 3 AAAGAAGAGA CCCTCCTAGA AAAGTAAAAT ATG ACT AAA AGC AAT GGAGAA GAG 54 Met Thr Lys Ser Asn Gly Glu Glu 1 5 CCC AAG ATG GGG GGC AGGATG GAG AGA TTC CAG CAG GGA GTC CGT AAA 102 Pro Lys Met Gly Gly Arg MetGlu Arg Phe Gln Gln Gly Val Arg Lys 10 15 20 CGC ACA CTT TTG GCC AAG AAGAAA GTG CAG AAC ATT ACA AAG GAG GTT 150 Arg Thr Leu Leu Ala Lys Lys LysVal Gln Asn Ile Thr Lys Glu Val 25 30 35 40 GTT AAA AGT TAC CTG TTT CGGAAT GCT TTT GTG CTG CTC ACA GTC ACC 198 Val Lys Ser Tyr Leu Phe Arg AsnAla Phe Val Leu Leu Thr Val Thr 45 50 55 GCT GTC ATT GTG GGT ACA ATC CTTGGA TTT ACC CTC CGA CCA TAC AGA 246 Ala Val Ile Val Gly Thr Ile Leu GlyPhe Thr Leu Arg Pro Tyr Arg 60 65 70 ATG AGC TAC CGG GAA GTC AAG TAC TTCTCC TTT CCT GGG GAA CTT CTG 294 Met Ser Tyr Arg Glu Val Lys Tyr Phe SerPhe Pro Gly Glu Leu Leu 75 80 85 ATG AGG ATG TTA CAG ATG CTG GTC TTA CCACTT ATC ATC TCC AGT CTT 342 Met Arg Met Leu Gln Met Leu Val Leu Pro LeuIle Ile Ser Ser Leu 90 95 100 GTC ACA GGA ATG GCG GCG CTA GAT AGT AAGGCA TCA GGG AAG TGG GAA 390 Val Thr Gly Met Ala Ala Leu Asp Ser Lys AlaSer Gly Lys Trp Glu 105 110 115 120 TGC GGA GCT GTA GTC TAT TAT ATG ACTACC ACC ATC ATT GCT GTG GTG 438 Cys Gly Ala Val Val Tyr Tyr Met Thr ThrThr Ile Ile Ala Val Val 125 130 135 ATT GGC ATA ATC ATT GTC ATC ATC ATCCAT CCT GGG AAG GGC ACA AAG 486 Ile Gly Ile Ile Ile Val Ile Ile Ile HisPro Gly Lys Gly Thr Lys 140 145 150 GAA AAC ATG CAC AGA GAA GGC AAA ATTGTA CGA GTG ACA GCT GCA GAT 534 Glu Asn Met His Arg Glu Gly Lys Ile ValArg Val Thr Ala Ala Asp 155 160 165 GCC TTC CTG GAC TTG ATC AGG AAC ATGTTA AAT CCA AAT CTG GTA GAA 582 Ala Phe Leu Asp Leu Ile Arg Asn Met LeuAsn Pro Asn Leu Val Glu 170 175 180 GCC TGC TTT AAA CAG TTT AAA ACC AACTAT GAG AAG AGA AGC TTT AAA 630 Ala Cys Phe Lys Gln Phe Lys Thr Asn TyrGlu Lys Arg Ser Phe Lys 185 190 195 200 GTG CCC ATC CAG GCC AAC GAA ACGCTT GTG GGT GCT GTG ATA AAC AAT 678 Val Pro Ile Gln Ala Asn Glu Thr LeuVal Gly Ala Val Ile Asn Asn 205 210 215 GTG TCT GAG GCC ATG GAG ACT CTTACC CGA ATC ACA GAG GAG CTG GTC 726 Val Ser Glu Ala Met Glu Thr Leu ThrArg Ile Thr Glu Glu Leu Val 220 225 230 CCA GTT CCA GGA TCT GTG AAT GGAGTC AAT GCC CTG GGT CTA GTT GTC 774 Pro Val Pro Gly Ser Val Asn Gly ValAsn Ala Leu Gly Leu Val Val 235 240 245 TTC TCC ATG TGC TTC GGT TTT GTGATT GGA AAC ATG AAG GAA CAG GGG 822 Phe Ser Met Cys Phe Gly Phe Val IleGly Asn Met Lys Glu Gln Gly 250 255 260 CAG GCC CTG AGA GAG TTC TTT GATTCT CTT AAC GAA GCC ATC ATG AGA 870 Gln Ala Leu Arg Glu Phe Phe Asp SerLeu Asn Glu Ala Ile Met Arg 265 270 275 280 CTG GTA GCA GTA ATA ATG TGGTAT GCC CCC GTG GGT ATT CTC TTC CTG 918 Leu Val Ala Val Ile Met Trp TyrAla Pro Val Gly Ile Leu Phe Leu 285 290 295 ATT GCT GGG AAG ATT GTG GAGATG GAA GAC ATG GGT GTG ATT GGG GGG 966 Ile Ala Gly Lys Ile Val Glu MetGlu Asp Met Gly Val Ile Gly Gly 300 305 310 CAG CTT GCC ATG TAC ACC GTGACT GTC ATT GTT GGC TTA CTC ATT CAC 1014 Gln Leu Ala Met Tyr Thr Val ThrVal Ile Val Gly Leu Leu Ile His 315 320 325 GCA GTC ATC GTC TTG CCA CTCCTC TAC TTC TTG GTA ACA CGG AAA AAC 1062 Ala Val Ile Val Leu Pro Leu LeuTyr Phe Leu Val Thr Arg Lys Asn 330 335 340 CCT TGG GTT TTT ATT GGA GGGTTG CTG CAA GCA CTC ATC ACC GCT CTG 1110 Pro Trp Val Phe Ile Gly Gly LeuLeu Gln Ala Leu Ile Thr Ala Leu 345 350 355 360 GGG ACC TCT TCA AGT TCTGCC ACC CTA CCC ATC ACC TTC AAG TGC CTG 1158 Gly Thr Ser Ser Ser Ser AlaThr Leu Pro Ile Thr Phe Lys Cys Leu 365 370 375 GAA GAG AAC AAT GGC GTGGAC AAG CGC GTC ACC AGA TTC GTG CTC CCC 1206 Glu Glu Asn Asn Gly Val AspLys Arg Val Thr Arg Phe Val Leu Pro 380 385 390 GTA GGA GCC ACC ATT AACATG GAT GGG ACT GCC CTC TAT GAG GCT TTG 1254 Val Gly Ala Thr Ile Asn MetAsp Gly Thr Ala Leu Tyr Glu Ala Leu 395 400 405 GCT GCC ATT TTC ATT GCTCAA GTT AAC AAC TTT GAA CTG AAC TTC GGA 1302 Ala Ala Ile Phe Ile Ala GlnVal Asn Asn Phe Glu Leu Asn Phe Gly 410 415 420 CAA ATT ATT ACA ATC AGCATC ACA GCC ACA GCT GCC AGT ATT GGG GCA 1350 Gln Ile Ile Thr Ile Ser IleThr Ala Thr Ala Ala Ser Ile Gly Ala 425 430 435 440 GCT GGA ATT CCT CAGGCG GGC CTG GTC ACT ATG GTC ATT GTG CTG ACA 1398 Ala Gly Ile Pro Gln AlaGly Leu Val Thr Met Val Ile Val Leu Thr 445 450 455 TCT GTC GGC CTG CCCACT GAC GAC ATC ACG CTC ATC ATC GCG GTG GAC 1446 Ser Val Gly Leu Pro ThrAsp Asp Ile Thr Leu Ile Ile Ala Val Asp 460 465 470 TGG TTC TTG GAT CGCCTC CGG ACC ACC ACC AAC GTA CTG GGA GAC TCC 1494 Trp Phe Leu Asp Arg LeuArg Thr Thr Thr Asn Val Leu Gly Asp Ser 475 480 485 CTG GGA GCT GGG ATTGTG GAG CAC TTG TCA CGA CAT GAA CTG AAG AAC 1542 Leu Gly Ala Gly Ile ValGlu His Leu Ser Arg His Glu Leu Lys Asn 490 495 500 AGA GAT GTT GAA ATGGGT AAC TCA GTG ATT GAA GAG AAT GAA ATG AAG 1590 Arg Asp Val Glu Met GlyAsn Ser Val Ile Glu Glu Asn Glu Met Lys 505 510 515 520 AAA CCA TAT CAACTG ATT GCA CAG GAC AAT GAA ACT GAG AAA CCC ATC 1638 Lys Pro Tyr Gln LeuIle Ala Gln Asp Asn Glu Thr Glu Lys Pro Ile 525 530 535 GAC AGT GAA ACCAAG ATG TAGACTAACA TAAAGAAACA CTTT 1680 Asp Ser Glu Thr Lys Met 540 542amino acids amino acid linear protein 4 Met Thr Lys Ser Asn Gly Glu GluPro Lys Met Gly Gly Arg Met Glu 1 5 10 15 Arg Phe Gln Gln Gly Val ArgLys Arg Thr Leu Leu Ala Lys Lys Lys 20 25 30 Val Gln Asn Ile Thr Lys GluVal Val Lys Ser Tyr Leu Phe Arg Asn 35 40 45 Ala Phe Val Leu Leu Thr ValThr Ala Val Ile Val Gly Thr Ile Leu 50 55 60 Gly Phe Thr Leu Arg Pro TyrArg Met Ser Tyr Arg Glu Val Lys Tyr 65 70 75 80 Phe Ser Phe Pro Gly GluLeu Leu Met Arg Met Leu Gln Met Leu Val 85 90 95 Leu Pro Leu Ile Ile SerSer Leu Val Thr Gly Met Ala Ala Leu Asp 100 105 110 Ser Lys Ala Ser GlyLys Trp Glu Cys Gly Ala Val Val Tyr Tyr Met 115 120 125 Thr Thr Thr IleIle Ala Val Val Ile Gly Ile Ile Ile Val Ile Ile 130 135 140 Ile His ProGly Lys Gly Thr Lys Glu Asn Met His Arg Glu Gly Lys 145 150 155 160 IleVal Arg Val Thr Ala Ala Asp Ala Phe Leu Asp Leu Ile Arg Asn 165 170 175Met Leu Asn Pro Asn Leu Val Glu Ala Cys Phe Lys Gln Phe Lys Thr 180 185190 Asn Tyr Glu Lys Arg Ser Phe Lys Val Pro Ile Gln Ala Asn Glu Thr 195200 205 Leu Val Gly Ala Val Ile Asn Asn Val Ser Glu Ala Met Glu Thr Leu210 215 220 Thr Arg Ile Thr Glu Glu Leu Val Pro Val Pro Gly Ser Val AsnGly 225 230 235 240 Val Asn Ala Leu Gly Leu Val Val Phe Ser Met Cys PheGly Phe Val 245 250 255 Ile Gly Asn Met Lys Glu Gln Gly Gln Ala Leu ArgGlu Phe Phe Asp 260 265 270 Ser Leu Asn Glu Ala Ile Met Arg Leu Val AlaVal Ile Met Trp Tyr 275 280 285 Ala Pro Val Gly Ile Leu Phe Leu Ile AlaGly Lys Ile Val Glu Met 290 295 300 Glu Asp Met Gly Val Ile Gly Gly GlnLeu Ala Met Tyr Thr Val Thr 305 310 315 320 Val Ile Val Gly Leu Leu IleHis Ala Val Ile Val Leu Pro Leu Leu 325 330 335 Tyr Phe Leu Val Thr ArgLys Asn Pro Trp Val Phe Ile Gly Gly Leu 340 345 350 Leu Gln Ala Leu IleThr Ala Leu Gly Thr Ser Ser Ser Ser Ala Thr 355 360 365 Leu Pro Ile ThrPhe Lys Cys Leu Glu Glu Asn Asn Gly Val Asp Lys 370 375 380 Arg Val ThrArg Phe Val Leu Pro Val Gly Ala Thr Ile Asn Met Asp 385 390 395 400 GlyThr Ala Leu Tyr Glu Ala Leu Ala Ala Ile Phe Ile Ala Gln Val 405 410 415Asn Asn Phe Glu Leu Asn Phe Gly Gln Ile Ile Thr Ile Ser Ile Thr 420 425430 Ala Thr Ala Ala Ser Ile Gly Ala Ala Gly Ile Pro Gln Ala Gly Leu 435440 445 Val Thr Met Val Ile Val Leu Thr Ser Val Gly Leu Pro Thr Asp Asp450 455 460 Ile Thr Leu Ile Ile Ala Val Asp Trp Phe Leu Asp Arg Leu ArgThr 465 470 475 480 Thr Thr Asn Val Leu Gly Asp Ser Leu Gly Ala Gly IleVal Glu His 485 490 495 Leu Ser Arg His Glu Leu Lys Asn Arg Asp Val GluMet Gly Asn Ser 500 505 510 Val Ile Glu Glu Asn Glu Met Lys Lys Pro TyrGln Leu Ile Ala Gln 515 520 525 Asp Asn Glu Thr Glu Lys Pro Ile Asp SerGlu Thr Lys Met 530 535 540 1800 base pairs nucleic acid single linearcDNA 5′UTR 1..33 CDS 34..1755 3′UTR 1756..1800 5 GATAGTGCTG AAGAGGAGGGGCGTTCCCAG ACC ATG GCA TCT ACG GAA GGT GCC 54 Met Ala Ser Thr Glu GlyAla 1 5 AAC AAT ATG CCC AAG CAG GTG GAA GTG CGA ATG CCA GAC AGT CAT CTT102 Asn Asn Met Pro Lys Gln Val Glu Val Arg Met Pro Asp Ser His Leu 1015 20 GGC TCA GAG GAA CCC AAG CAC CGG CAC CTG GGC CTG CGC CTG TGT GAC150 Gly Ser Glu Glu Pro Lys His Arg His Leu Gly Leu Arg Leu Cys Asp 2530 35 AAG CTG GGG AAG AAT CTG CTG CTC ACC CTG ACG GTG TTT GGT GTC ATC198 Lys Leu Gly Lys Asn Leu Leu Leu Thr Leu Thr Val Phe Gly Val Ile 4045 50 55 CTG GGA GCA GTG TGT GGA GGG CTT CTT CGC TTG GCA TCT CCC ATC CAC246 Leu Gly Ala Val Cys Gly Gly Leu Leu Arg Leu Ala Ser Pro Ile His 6065 70 CCT GAT GTG GTT ATG TTA ATA GCC TTC CCA GGG GAT ATA CTC ATG AGG294 Pro Asp Val Val Met Leu Ile Ala Phe Pro Gly Asp Ile Leu Met Arg 7580 85 ATG CTA AAA ATG CTC ATT CTG GGT CTA ATC ATC TCC AGC TTA ATC ACA342 Met Leu Lys Met Leu Ile Leu Gly Leu Ile Ile Ser Ser Leu Ile Thr 9095 100 GGG TTG TCA GGC CTG GAT GCT AAG GCT AGT GGC CGC TTG GGC ACG AGA390 Gly Leu Ser Gly Leu Asp Ala Lys Ala Ser Gly Arg Leu Gly Thr Arg 105110 115 GCC ATG GTG TAT TAC ATG TCC ACG ACC ATC ATT GCT GCA GTA CTG GGG438 Ala Met Val Tyr Tyr Met Ser Thr Thr Ile Ile Ala Ala Val Leu Gly 120125 130 135 GTC ATT CTG GTC TTG GCT ATC CAT CCA GGC AAT CCC AAG CTC AAGAAG 486 Val Ile Leu Val Leu Ala Ile His Pro Gly Asn Pro Lys Leu Lys Lys140 145 150 CAG CTG GGG CCT GGG AAG AAG AAT GAT GAA GTG TCC AGC CTG GATGCC 534 Gln Leu Gly Pro Gly Lys Lys Asn Asp Glu Val Ser Ser Leu Asp Ala155 160 165 TTC CTG GAC CTT ATT CGA AAT CTC TTC CCT GAA AAC CTT GTC CAAGCC 582 Phe Leu Asp Leu Ile Arg Asn Leu Phe Pro Glu Asn Leu Val Gln Ala170 175 180 TGC TTT CAA CAG ATT CAA ACA GTG ACG AAG AAA GTC CTG GTT GCACCA 630 Cys Phe Gln Gln Ile Gln Thr Val Thr Lys Lys Val Leu Val Ala Pro185 190 195 CCG CCA GAC GAG GAG GCC AAC GCA ACC AGC GCT GAA GTC TCT CTGTTG 678 Pro Pro Asp Glu Glu Ala Asn Ala Thr Ser Ala Glu Val Ser Leu Leu200 205 210 215 AAC GAG ACT GTG ACT GAG GTG CCG GAG GAG ACT AAG ATG GTTATC AAG 726 Asn Glu Thr Val Thr Glu Val Pro Glu Glu Thr Lys Met Val IleLys 220 225 230 AAG GGC CTG GAG TTC AAG GAT GGG ATG AAC GTC TTA GGT CTGATA GGG 774 Lys Gly Leu Glu Phe Lys Asp Gly Met Asn Val Leu Gly Leu IleGly 235 240 245 TTT TTC ATT GCT TTT GGC ATC GCT ATG GGG AAG ATG GGA GATCAG GCC 822 Phe Phe Ile Ala Phe Gly Ile Ala Met Gly Lys Met Gly Asp GlnAla 250 255 260 AAG CTG ATG GTG GAT TTC TTC AAC ATT TTG AAT GAG ATT GTAATG AAG 870 Lys Leu Met Val Asp Phe Phe Asn Ile Leu Asn Glu Ile Val MetLys 265 270 275 TTA GTG ATC ATG ATC ATG TGG TAC TCT CCC CTG GGT ATC GCCTGC CTG 918 Leu Val Ile Met Ile Met Trp Tyr Ser Pro Leu Gly Ile Ala CysLeu 280 285 290 295 ATC TGT GGA AAG ATC ATT GCA ATC AAG GAC TTA GAA GTGGTT GCT AGG 966 Ile Cys Gly Lys Ile Ile Ala Ile Lys Asp Leu Glu Val ValAla Arg 300 305 310 CAA CTG GGG ATG TAC ATG GTA ACA GTG ATC ATA GGC CTCATC ATC CAC 1014 Gln Leu Gly Met Tyr Met Val Thr Val Ile Ile Gly Leu IleIle His 315 320 325 GGG GGC ATC TTT CTC CCC TTG ATT TAC TTT GTA GTG ACCAGG AAA AAC 1062 Gly Gly Ile Phe Leu Pro Leu Ile Tyr Phe Val Val Thr ArgLys Asn 330 335 340 CCC TTC TCC CTT TTT GCT GGC ATT TTC CAA GCT TGG ATCACT GCC CTG 1110 Pro Phe Ser Leu Phe Ala Gly Ile Phe Gln Ala Trp Ile ThrAla Leu 345 350 355 GGC ACC GCT TCC AGT GCT GGA ACT TTG CCT GTC ACC TTTCGT TGC CTG 1158 Gly Thr Ala Ser Ser Ala Gly Thr Leu Pro Val Thr Phe ArgCys Leu 360 365 370 375 GAA GAA AAT CTG GGG ATT GAT AAG CGT GTG ACT AGATTC GTC CTT CCT 1206 Glu Glu Asn Leu Gly Ile Asp Lys Arg Val Thr Arg PheVal Leu Pro 380 385 390 GTT GGA GCA ACC ATT AAC ATG GAT GGT ACA GCC CTTTAT GAA GCG GTG 1254 Val Gly Ala Thr Ile Asn Met Asp Gly Thr Ala Leu TyrGlu Ala Val 395 400 405 GCC GCC ATC TTT ATA GCC CAA ATG AAT GGT GTT GTCCTG GAT GGA GGA 1302 Ala Ala Ile Phe Ile Ala Gln Met Asn Gly Val Val LeuAsp Gly Gly 410 415 420 CAG ATT GTG ACT GTA AGC CTC ACA GCC ACC CTG GCAAGC GTC GGC GCG 1350 Gln Ile Val Thr Val Ser Leu Thr Ala Thr Leu Ala SerVal Gly Ala 425 430 435 GCC AGT ATC CCC AGT GCC GGG CTG GTC ACC ATG CTCCTC ATT CTG ACA 1398 Ala Ser Ile Pro Ser Ala Gly Leu Val Thr Met Leu LeuIle Leu Thr 440 445 450 455 GCC GTG GGC CTG CCA ACA GAG GAC ATC AGC TTGCTG GTG GCT GTG GAC 1446 Ala Val Gly Leu Pro Thr Glu Asp Ile Ser Leu LeuVal Ala Val Asp 460 465 470 TGG CTG CTG GAC AGG ATG AGA ACT TCA GTC AATGTT GTG GGT GAC TCT 1494 Trp Leu Leu Asp Arg Met Arg Thr Ser Val Asn ValVal Gly Asp Ser 475 480 485 TTT GGG GCT GGG ATA GTC TAT CAC CTC TCC AAGTCT GAG CTG GAT ACC 1542 Phe Gly Ala Gly Ile Val Tyr His Leu Ser Lys SerGlu Leu Asp Thr 490 495 500 ATT GAC TCC CAG CAT CGA GTG CAT GAA GAT ATTGAA ATG ACC AAG ACT 1590 Ile Asp Ser Gln His Arg Val His Glu Asp Ile GluMet Thr Lys Thr 505 510 515 CAA TCC ATT TAT GAT GAC ATG AAG AAC CAC AGGGAA AGC AAC TCT AAT 1638 Gln Ser Ile Tyr Asp Asp Met Lys Asn His Arg GluSer Asn Ser Asn 520 525 530 535 CAA TGT GTC TAT GCT GCA CAC AAC TCT GTCATA GTA GAT GAA TGC AAG 1686 Gln Cys Val Tyr Ala Ala His Asn Ser Val IleVal Asp Glu Cys Lys 540 545 550 GTA ACT CTG GCA GCC AAT GGA AAG TCA GCCGAC TGC AGT GTT GAG GAA 1734 Val Thr Leu Ala Ala Asn Gly Lys Ser Ala AspCys Ser Val Glu Glu 555 560 565 GAA CCT TGG AAA CGT GAG AAA TAAGGATATGAGTCTCAGCA AATTCTTGAA 1785 Glu Pro Trp Lys Arg Glu Lys 570 TAAACTCCCCAGCGT 1800 574 amino acids amino acid linear protein 6 Met Ala Ser ThrGlu Gly Ala Asn Asn Met Pro Lys Gln Val Glu Val 1 5 10 15 Arg Met ProAsp Ser His Leu Gly Ser Glu Glu Pro Lys His Arg His 20 25 30 Leu Gly LeuArg Leu Cys Asp Lys Leu Gly Lys Asn Leu Leu Leu Thr 35 40 45 Leu Thr ValPhe Gly Val Ile Leu Gly Ala Val Cys Gly Gly Leu Leu 50 55 60 Arg Leu AlaSer Pro Ile His Pro Asp Val Val Met Leu Ile Ala Phe 65 70 75 80 Pro GlyAsp Ile Leu Met Arg Met Leu Lys Met Leu Ile Leu Gly Leu 85 90 95 Ile IleSer Ser Leu Ile Thr Gly Leu Ser Gly Leu Asp Ala Lys Ala 100 105 110 SerGly Arg Leu Gly Thr Arg Ala Met Val Tyr Tyr Met Ser Thr Thr 115 120 125Ile Ile Ala Ala Val Leu Gly Val Ile Leu Val Leu Ala Ile His Pro 130 135140 Gly Asn Pro Lys Leu Lys Lys Gln Leu Gly Pro Gly Lys Lys Asn Asp 145150 155 160 Glu Val Ser Ser Leu Asp Ala Phe Leu Asp Leu Ile Arg Asn LeuPhe 165 170 175 Pro Glu Asn Leu Val Gln Ala Cys Phe Gln Gln Ile Gln ThrVal Thr 180 185 190 Lys Lys Val Leu Val Ala Pro Pro Pro Asp Glu Glu AlaAsn Ala Thr 195 200 205 Ser Ala Glu Val Ser Leu Leu Asn Glu Thr Val ThrGlu Val Pro Glu 210 215 220 Glu Thr Lys Met Val Ile Lys Lys Gly Leu GluPhe Lys Asp Gly Met 225 230 235 240 Asn Val Leu Gly Leu Ile Gly Phe PheIle Ala Phe Gly Ile Ala Met 245 250 255 Gly Lys Met Gly Asp Gln Ala LysLeu Met Val Asp Phe Phe Asn Ile 260 265 270 Leu Asn Glu Ile Val Met LysLeu Val Ile Met Ile Met Trp Tyr Ser 275 280 285 Pro Leu Gly Ile Ala CysLeu Ile Cys Gly Lys Ile Ile Ala Ile Lys 290 295 300 Asp Leu Glu Val ValAla Arg Gln Leu Gly Met Tyr Met Val Thr Val 305 310 315 320 Ile Ile GlyLeu Ile Ile His Gly Gly Ile Phe Leu Pro Leu Ile Tyr 325 330 335 Phe ValVal Thr Arg Lys Asn Pro Phe Ser Leu Phe Ala Gly Ile Phe 340 345 350 GlnAla Trp Ile Thr Ala Leu Gly Thr Ala Ser Ser Ala Gly Thr Leu 355 360 365Pro Val Thr Phe Arg Cys Leu Glu Glu Asn Leu Gly Ile Asp Lys Arg 370 375380 Val Thr Arg Phe Val Leu Pro Val Gly Ala Thr Ile Asn Met Asp Gly 385390 395 400 Thr Ala Leu Tyr Glu Ala Val Ala Ala Ile Phe Ile Ala Gln MetAsn 405 410 415 Gly Val Val Leu Asp Gly Gly Gln Ile Val Thr Val Ser LeuThr Ala 420 425 430 Thr Leu Ala Ser Val Gly Ala Ala Ser Ile Pro Ser AlaGly Leu Val 435 440 445 Thr Met Leu Leu Ile Leu Thr Ala Val Gly Leu ProThr Glu Asp Ile 450 455 460 Ser Leu Leu Val Ala Val Asp Trp Leu Leu AspArg Met Arg Thr Ser 465 470 475 480 Val Asn Val Val Gly Asp Ser Phe GlyAla Gly Ile Val Tyr His Leu 485 490 495 Ser Lys Ser Glu Leu Asp Thr IleAsp Ser Gln His Arg Val His Glu 500 505 510 Asp Ile Glu Met Thr Lys ThrGln Ser Ile Tyr Asp Asp Met Lys Asn 515 520 525 His Arg Glu Ser Asn SerAsn Gln Cys Val Tyr Ala Ala His Asn Ser 530 535 540 Val Ile Val Asp GluCys Lys Val Thr Leu Ala Ala Asn Gly Lys Ser 545 550 555 560 Ala Asp CysSer Val Glu Glu Glu Pro Trp Lys Arg Glu Lys 565 570 1674 base pairsnucleic acid single linear cDNA 5′UTR 1..15 CDS 16..1590 3′UTR1591..1674 7 ATAGCGGCGA CAGCC ATG GGG AAA CCG GCG AGG AAA GGA TGC CCGAGT TGG 51 Met Gly Lys Pro Ala Arg Lys Gly Cys Pro Ser Trp 1 5 10 AAGCGC TTC CTG AAG AAT AAC TGG GTG TTG CTG TCC ACC GTG GCC GCG 99 Lys ArgPhe Leu Lys Asn Asn Trp Val Leu Leu Ser Thr Val Ala Ala 15 20 25 GTG GTGCTA GGC ATT ACC ACA GGA GTC TTG GTT CGA GAA CAC AGC AAC 147 Val Val LeuGly Ile Thr Thr Gly Val Leu Val Arg Glu His Ser Asn 30 35 40 CTC TCA ACTCTA GAG AAA TTC TAC TTT GCT TTT CCT GGA GAA ATT CTA 195 Leu Ser Thr LeuGlu Lys Phe Tyr Phe Ala Phe Pro Gly Glu Ile Leu 45 50 55 60 ATG CGG ATGCTG AAA CTC ATC ATT TTG CCA TTA ATT ATA TCC AGC ATG 243 Met Arg Met LeuLys Leu Ile Ile Leu Pro Leu Ile Ile Ser Ser Met 65 70 75 ATT ACA GGT GTTGCT GCA CTG GAT TCC AAC GTA TCC GGA AAA ATT GGT 291 Ile Thr Gly Val AlaAla Leu Asp Ser Asn Val Ser Gly Lys Ile Gly 80 85 90 CTG CGC GCT GTC GTGTAT TAT TTC TGT ACC ACT CTC ATT GCT GTT ATT 339 Leu Arg Ala Val Val TyrTyr Phe Cys Thr Thr Leu Ile Ala Val Ile 95 100 105 CTA GGT ATT GTG CTGGTG GTG AGC ATC AAG CCT GGT GTC ACC CAG AAA 387 Leu Gly Ile Val Leu ValVal Ser Ile Lys Pro Gly Val Thr Gln Lys 110 115 120 GTG GGT GAA ATT GCGAGG ACA GGC AGC ACC CCT GAA GTC AGT ACG GTG 435 Val Gly Glu Ile Ala ArgThr Gly Ser Thr Pro Glu Val Ser Thr Val 125 130 135 140 GAT GCC ATG TTAGAT CTC ATC AGG AAT ATG TTC CCT GAG AAT CTT GTC 483 Asp Ala Met Leu AspLeu Ile Arg Asn Met Phe Pro Glu Asn Leu Val 145 150 155 CAG GCC TGT TTTCAG CAG TAC AAA ACT AAG CGT GAA GAA GTG AAG CCT 531 Gln Ala Cys Phe GlnGln Tyr Lys Thr Lys Arg Glu Glu Val Lys Pro 160 165 170 CCC AGC GAT CCAGAG ATG AAC ATG ACA GAA GAG TCC TTC ACA GCT GTC 579 Pro Ser Asp Pro GluMet Asn Met Thr Glu Glu Ser Phe Thr Ala Val 175 180 185 ATG ACA ACT GCAATT TCC AAG AAC AAA ACA AAG GAA TAC AAA ATT GTT 627 Met Thr Thr Ala IleSer Lys Asn Lys Thr Lys Glu Tyr Lys Ile Val 190 195 200 GGC ATG TAT TCAGAT GGC ATA AAC GTC CTG GGC TTG ATT GTC TTT TGC 675 Gly Met Tyr Ser AspGly Ile Asn Val Leu Gly Leu Ile Val Phe Cys 205 210 215 220 CTT GTC TTTGGA CTT GTC ATT GGA AAA ATG GGA GAA AAG GGA CAA ATT 723 Leu Val Phe GlyLeu Val Ile Gly Lys Met Gly Glu Lys Gly Gln Ile 225 230 235 CTG GTG GATTTC TTC AAT GCT TTG AGT GAT GCA ACC ATG AAA ATC GTT 771 Leu Val Asp PhePhe Asn Ala Leu Ser Asp Ala Thr Met Lys Ile Val 240 245 250 CAG ATC ATCATG TGT TAT ATG CCA CTA GGT ATT TTG TTC CTG ATT GCT 819 Gln Ile Ile MetCys Tyr Met Pro Leu Gly Ile Leu Phe Leu Ile Ala 255 260 265 GGG AAG ATCATA GAA GTT GAA GAC TGG GAA ATA TTC CGC AAG CTG GGC 867 Gly Lys Ile IleGlu Val Glu Asp Trp Glu Ile Phe Arg Lys Leu Gly 270 275 280 CTT TAC ATGGCC ACA GTC CTG ACT GGG CTT GCA ATC CAC TCC ATT GTA 915 Leu Tyr Met AlaThr Val Leu Thr Gly Leu Ala Ile His Ser Ile Val 285 290 295 300 ATT CTCCCG CTG ATA TAT TTC ATA GTC GTA CGA AAG AAC CCT TTC CGA 963 Ile Leu ProLeu Ile Tyr Phe Ile Val Val Arg Lys Asn Pro Phe Arg 305 310 315 TTT GCCATG GGA ATG GCC CAG GCT CTC CTG ACA GCT CTC ATG ATC TCT 1011 Phe Ala MetGly Met Ala Gln Ala Leu Leu Thr Ala Leu Met Ile Ser 320 325 330 TCC AGTTCA GCA ACA CTG CCT GTC ACC TTC CGC TGT GCT GAA GAA AAT 1059 Ser Ser SerAla Thr Leu Pro Val Thr Phe Arg Cys Ala Glu Glu Asn 335 340 345 AAC CAGGTG GAC AAG AGG ATC ACT CGA TTC GTG TTA CCC GTT GGT GCA 1107 Asn Gln ValAsp Lys Arg Ile Thr Arg Phe Val Leu Pro Val Gly Ala 350 355 360 ACA ATCAAC ATG GAT GGG ACC GCG CTC TAT GAA GCA GTG GCA GCG GTG 1155 Thr Ile AsnMet Asp Gly Thr Ala Leu Tyr Glu Ala Val Ala Ala Val 365 370 375 380 TTTATT GCA CAG TTG AAT GAC CTG GAC TTG GGC ATT GGG CAG ATC ATC 1203 Phe IleAla Gln Leu Asn Asp Leu Asp Leu Gly Ile Gly Gln Ile Ile 385 390 395 ACCATC AGT ATC ACG GCC ACA TCT GCC AGC ATC GGA GCT GCT GGC GTG 1251 Thr IleSer Ile Thr Ala Thr Ser Ala Ser Ile Gly Ala Ala Gly Val 400 405 410 CCCCAG GCT GGC CTG GTG ACC ATG GTG ATT GTG CTG AGT GCC GTG GGC 1299 Pro GlnAla Gly Leu Val Thr Met Val Ile Val Leu Ser Ala Val Gly 415 420 425 CTGCCC GCC GAG GAT GTC ACC CTG ATC ATT GCT GTC GAC TGG CTC CTG 1347 Leu ProAla Glu Asp Val Thr Leu Ile Ile Ala Val Asp Trp Leu Leu 430 435 440 GACCGG TTC AGG ACC ATG GTC AAC GTC CTT GGT GAT GCT TTT GGG ACG 1395 Asp ArgPhe Arg Thr Met Val Asn Val Leu Gly Asp Ala Phe Gly Thr 445 450 455 460GGC ATT GTG GAA AAG CTC TCC AAG AAG GAG CTG GAG CAG ATG GAT GTT 1443 GlyIle Val Glu Lys Leu Ser Lys Lys Glu Leu Glu Gln Met Asp Val 465 470 475TCA TCT GAA GTC AAC ATT GTG AAT CCC TTT GCC TTG GAA TCC ACA ATC 1491 SerSer Glu Val Asn Ile Val Asn Pro Phe Ala Leu Glu Ser Thr Ile 480 485 490CTT GAC AAC GAA GAC TCA GAC ACC AAG AAG TCT TAT GTC AAT GGA GGC 1539 LeuAsp Asn Glu Asp Ser Asp Thr Lys Lys Ser Tyr Val Asn Gly Gly 495 500 505TTT GCA GTA GAC AAG TCT GAC ACC ATC TCA TTC ACC CAG ACC TCA CAG 1587 PheAla Val Asp Lys Ser Asp Thr Ile Ser Phe Thr Gln Thr Ser Gln 510 515 520TTC TAGGGCCCCT GGCTGCAGAT GACTGGAAAC AAGGAAGGAC ATTTCGTGAG 1640 Phe 525AGTCATCTCA AACACGGCTT AAGGAAAAGA GAAA 1674 525 amino acids amino acidlinear protein 8 Met Gly Lys Pro Ala Arg Lys Gly Cys Pro Ser Trp Lys ArgPhe Leu 1 5 10 15 Lys Asn Asn Trp Val Leu Leu Ser Thr Val Ala Ala ValVal Leu Gly 20 25 30 Ile Thr Thr Gly Val Leu Val Arg Glu His Ser Asn LeuSer Thr Leu 35 40 45 Glu Lys Phe Tyr Phe Ala Phe Pro Gly Glu Ile Leu MetArg Met Leu 50 55 60 Lys Leu Ile Ile Leu Pro Leu Ile Ile Ser Ser Met IleThr Gly Val 65 70 75 80 Ala Ala Leu Asp Ser Asn Val Ser Gly Lys Ile GlyLeu Arg Ala Val 85 90 95 Val Tyr Tyr Phe Cys Thr Thr Leu Ile Ala Val IleLeu Gly Ile Val 100 105 110 Leu Val Val Ser Ile Lys Pro Gly Val Thr GlnLys Val Gly Glu Ile 115 120 125 Ala Arg Thr Gly Ser Thr Pro Glu Val SerThr Val Asp Ala Met Leu 130 135 140 Asp Leu Ile Arg Asn Met Phe Pro GluAsn Leu Val Gln Ala Cys Phe 145 150 155 160 Gln Gln Tyr Lys Thr Lys ArgGlu Glu Val Lys Pro Pro Ser Asp Pro 165 170 175 Glu Met Asn Met Thr GluGlu Ser Phe Thr Ala Val Met Thr Thr Ala 180 185 190 Ile Ser Lys Asn LysThr Lys Glu Tyr Lys Ile Val Gly Met Tyr Ser 195 200 205 Asp Gly Ile AsnVal Leu Gly Leu Ile Val Phe Cys Leu Val Phe Gly 210 215 220 Leu Val IleGly Lys Met Gly Glu Lys Gly Gln Ile Leu Val Asp Phe 225 230 235 240 PheAsn Ala Leu Ser Asp Ala Thr Met Lys Ile Val Gln Ile Ile Met 245 250 255Cys Tyr Met Pro Leu Gly Ile Leu Phe Leu Ile Ala Gly Lys Ile Ile 260 265270 Glu Val Glu Asp Trp Glu Ile Phe Arg Lys Leu Gly Leu Tyr Met Ala 275280 285 Thr Val Leu Thr Gly Leu Ala Ile His Ser Ile Val Ile Leu Pro Leu290 295 300 Ile Tyr Phe Ile Val Val Arg Lys Asn Pro Phe Arg Phe Ala MetGly 305 310 315 320 Met Ala Gln Ala Leu Leu Thr Ala Leu Met Ile Ser SerSer Ser Ala 325 330 335 Thr Leu Pro Val Thr Phe Arg Cys Ala Glu Glu AsnAsn Gln Val Asp 340 345 350 Lys Arg Ile Thr Arg Phe Val Leu Pro Val GlyAla Thr Ile Asn Met 355 360 365 Asp Gly Thr Ala Leu Tyr Glu Ala Val AlaAla Val Phe Ile Ala Gln 370 375 380 Leu Asn Asp Leu Asp Leu Gly Ile GlyGln Ile Ile Thr Ile Ser Ile 385 390 395 400 Thr Ala Thr Ser Ala Ser IleGly Ala Ala Gly Val Pro Gln Ala Gly 405 410 415 Leu Val Thr Met Val IleVal Leu Ser Ala Val Gly Leu Pro Ala Glu 420 425 430 Asp Val Thr Leu IleIle Ala Val Asp Trp Leu Leu Asp Arg Phe Arg 435 440 445 Thr Met Val AsnVal Leu Gly Asp Ala Phe Gly Thr Gly Ile Val Glu 450 455 460 Lys Leu SerLys Lys Glu Leu Glu Gln Met Asp Val Ser Ser Glu Val 465 470 475 480 AsnIle Val Asn Pro Phe Ala Leu Glu Ser Thr Ile Leu Asp Asn Glu 485 490 495Asp Ser Asp Thr Lys Lys Ser Tyr Val Asn Gly Gly Phe Ala Val Asp 500 505510 Lys Ser Asp Thr Ile Ser Phe Thr Gln Thr Ser Gln Phe 515 520 525 31base pairs nucleic acid single linear cDNA 9 CGCGGGTACC AAYCTSGTVSARGCYTGYTT Y 31 31 base pairs nucleic acid single linear cDNA 10CGCGTCTAGA YTGDGCDATR AARAYBGCDG C 31 63 base pairs nucleic acid singlelinear cDNA 11 CTGRGCRATG AARATGGCAG CCAGGGCYTC ATACAGGGCT GTGCCRTCCA 50TGTTRATGGT RGC 63 34 base pairs nucleic acid single linear cDNA 12GCGCGTCGAC AAGCTTGCCA TGCAACAGCC TGTT 34 28 base pairs nucleic acidsingle linear cDNA 13 GCGCTCTAGA TCAGCCCACG GTCAGTTG 28

We claim:
 1. An antibody or fragment thereof that is immunologicallyreactive to a human excitatory amino acid transporter that is EAAT4. 2.The antibody or fragment thereof according to claim 1, wherein theantibody is a monoclonal antibody.
 3. The antibody or fragment thereofaccording to claim 1, wherein a human excitatory amino acid transporterhas an amino acid sequence identified by Seq. ID No.
 2. 4. A cell linewhich produces an antibody or fragment thereof that is immunologicallyreactive to a human excitatory amino acid transporter that is EAAT4. 5.The cell line according to claim 4, wherein the antibody is a monoclonalantibody.
 6. The cell line according to claim 4, wherein a humanexcitatory amino acid transporter has an amino acid sequence identifiedby Seq. ID No.
 2. 7. An epitope of a human excitatory amino acidtransporter that is EAAT4 wherein the epitope is immunologicallyreactive to the antibody or fragment thereof according to claim
 1. 8.The epitope according to claim 7 wherein a human excitatory amino acidtransporter has an amino acid sequence identified by Seq. ID No. 2.